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Design and combinatorial synthesis approach of non-peptidic trimeric small molecules mimicking i, i + 4(3), i + 7 positi...

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Design and combinatorial synthesis approach of non-peptidic trimeric small molecules mimicking i, i + 4(3), i + 7 positions of alpha-helices
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English
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Zhou, Mingzhou
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University of South Florida
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Protein-protein interaction
Mimetics
Alpha-helical
Non-peptidic
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Protein-protein interactions are key to several biological processes that facilitate signal transduction and many other processes. These interactions are involved in pathways that are critical to many human diseases. Targeting specific protein-protein interactions is a challenging goal because protein-protein interactions are predominately through hydrophobic interactions. Antagonists of the protein-protein interactions need to be perfectly fit into the binding pockets to ensure the activity. The alpha-helical domain of the proteins behaves as the recognition motifs for numerous protein-protein, and protein-nucleic acid interactions. Research has shown that pathways of many diseases contain protein-protein interactions involving alpha-helical domains, e.g. neurological disorders, bacterial infections, HIV and cancer, etc. It is difficult yet very important to design small molecules to target the shallow binding areas of protein-protein interactions. So far the most successful one is Hamilton's 1,4-terphenylene scaffold, which has been used to target the interactions between p53/MDM2, Bak/Bcl-xL etc. Inspired by this, we designed and synthesized three new scaffolds of non-pepditic alpha-helical mimetics, mimicking the i, i + 4, i + 7 positions of an alpha-helix. There are three basic principles that were leading our design. The side chains of our designed molecules should act as mimetics of the side chains of an alpha-helix. Second, our molecules should possess improved water solubility. Third, the molecules should be easy to synthesize to generate a focused library. Some of our molecules, including the ones whose molecular weight are as low as 294, started to show some inhibition against p53/MDM2 interactions.
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Dissertation (PHD)--University of South Florida, 2010.
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by Mingzhou Zhou.
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Design and Combinatorial Synthesis Approach of Non peptidic Trimeric Small Molecules Mimicking i i +4(3), i +7 Positions of Helices by M ingzhou Zhou A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Mark L. Mclaughlin, Ph.D. Jianfeng Cai Ph.D. Wayne C. Guida, Ph.D. Rongshi Li Ph.D. Roman Manetsch, Ph.D. Date of Approval: July 6 2010 Keywords: protein protein interaction, mimetics, helical, non peptidic Copyright 2010, Mingzhou Zhou

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The search for truth has shattered most of my old beliefs and has made me commit what are probably sins where otherwise I should have kept clear of them. I do not think it has in any way made me happier; of course it has given me a deeper character, a contempt for trifles or mockery, but at the same time it has taken away cheerfulness and made it much harder to make bosom fri ends and, worst of all, it has debarred me from free intercourse with my people, and thus made them strangers to some of my deepest thoughts which, if by any mischance I do let them out, immediately become the subject for mockery which is inexpressively bi tter to me though not unkindly meant. -Bertrand Arthur William Russell

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Acknowledgement s First and foremost, I would like to thank Dr. Mark L. McLaughlin for his superior instruction du ring my Ph.D. studies. Dr. McLaughlin not only led me to solve problems during my research, but, more importantly to be a professional researcher. I would also like to thank current and previous committee members Dr. Wayne C. Guida, Dr. Roman Manetsch, Dr. Rongshi Li, Dr. Jianfeng Cai, and Dr. Peter Zhang for providing invaluable advice during my learning process. I would like to thank curren t and previous group members Dr. Missy Topper, Hyun Joo Kil, Dr. Priyesh Jain, David Badger, Dr. Laura Anderson, Yi Liang, Fenger Zhou, Sridhar Kaulagari, Dr. Vasudha Sharma, Dr. Umut Oguz, and Dr. Sung Wook Yi. A positive and supportive atmosphere is an important factor leading to efficient research. I would like to thank Dr the ELISA essay, Daniel N. Santiago for performing the computational study and Dr. Huiling Jiang for synthesizing some reagents for my research Furthermore, I would like to thank Dr. Daniele Pernazza and Dr. Roberta Pireddu for providing numerous suggestions for my dissertation. They as well as Dr. Nick Lawrence, Dr. Harshani Lawrence, Yunting Luo, Dr. Yiyu Ge, and Dr. Binglin Wang helped m e prepare for my oral defense for my dissertation. I would like to send speci al thanks to Kelly M. Carper for all the grammar correction s of my dissertation.

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Lastly, I would like to thank the NIH NCI P01 CA118210 for financial support of the work described in this dissertation that was carried out at Moffitt Cancer Center and U nive rsity of S outh F lorida

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i Table of Contents List of Tables ................................ ................................ ................................ ...................... v List of Figures ................................ ................................ ................................ .................... v i List of Schemes ................................ ................................ ................................ .................. i x List of Abbreviations ................................ ................................ ................................ ......... x i Abstract ................................ ................................ ................................ ............................ x i i i Chapter One : Peptidic and Non peptidic Scaffolds to Disrupt Protein protein Interactions that Involve Helices ................................ ............................... 1 1. 1 Protein protein interaction s ................................ ................................ ................ 1 1.2 Efforts to target the protein protein interaction ................................ ................. 1 1.3 Efforts to target the protein protein interactions involving helix .................. 2 1.4 Peptidic Helical mimetics ................................ ................................ .............. 3 1. 4 1 Cyclic peptides ................................ ................................ .................... 4 1. 4 2 Direct covalent bonding of the side chains of the amino acids ........... 4 1. 4 3 Metal coordinate bonding ................................ ................................ ... 6 1. 4 4 Connecting side chains of unnatural amino acids at i and i + 7 positions ................................ ................................ ............................. 7 1. 4 5 Connecting i and i + 7 side chains with a photo controlled azobenzene linker ................................ ................................ ................ 8 1. 5 Miniproteins ................................ ................................ ................................ ....... 9 1.6 Foldamers ................................ ................................ ................................ ......... 1 0 1.7 Synthetic non peptidic helical mimetics with linear scaffold ...................... 1 1 1.7.1 Helical mimetic scaf folds developed by Hamilton and coworkers ................................ ................................ .......................... 1 4 1.7.2 Helical mimetic scaffolds developed by Rebek and coworkers ................................ ................................ .......................... 16 1.7.3 Trisubstituted imidazole scaffold ................................ ...................... 17 1.7.4 Benzylideneacetophenone scaffold ................................ ................... 1 8 1.7.5 trans Fused polycylic ethers scaffold ................................ ............... 19 1.8 Synthetic non peptidic helical mimetics with other scaffold ....................... 20 1.8.1 Nutlin ................................ ................................ ................................ 20 1.8.2 1,4 Benzodiazepine 2,5 dione scaffolds ................................ ........... 21 1.8.3 Coactivator binding inhibitors ................................ .......................... 22

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ii 1.9 Summary of non peptidic helical mimetics ................................ ................. 23 1.10 Possibility of design general non peptidic hel ical scaffold ....................... 2 3 1.11 Balance between rational design and combinatorial chemistry ..................... 2 5 1.12 Cancer: our target ................................ ................................ ........................... 26 1.12.1 Hallmarks of cancer ................................ ................................ ........ 26 1.12.2 Apoptosis ................................ ................................ ........................ 2 7 1.12.3 p53/MDM2 interaction ................................ ................................ ... 28 1.12.4 Bcl 2 family protein protein interactions ................................ ........ 3 0 1.13 References ................................ ................................ ................................ ...... 31 Chapter Two: Design and Synthesis of 2,5 Terpyrimidinylenes as More Drug l ike 1,4 Terphenylene Mimetics ................................ ................................ 36 2.1 Introduction ................................ ................................ ................................ ...... 36 2.1.1 Terphenylene Scaffold and Related Work ............... 36 2.1.2 Design of the 2,5 terpyrimidinylene scaffold ................................ ... 39 2.2 Results and Discussion for the 1,4 pyrimidinylene scaffold ........................... 40 2.2.1 Retrosynthesis of the 1,4 pyrimidinylene scaffold ........................... 40 2.2.2 Synthesis of pyrimidine monomer 2.4 library ................................ .. 41 2.2.3 Conversion of the 5 cyano group to an amidine ............................... 43 2.2.4 Synthesis of the pyrimidine dimer ................................ .................... 45 2.2.5 X Ray crystal structure of a representative dimer ............................ 46 2.2.6 Synthesis of the pyrimidine trimers ................................ .................. 47 2.3 Conclusion ................................ ................................ ................................ ....... 48 2.4 Experimental Section ................................ ................................ ....................... 48 2.4.1 Materials and Methods ................................ ................................ ...... 48 2.4.2 Experimental Procedures ................................ ................................ .. 49 2.5 References ................................ ................................ ................................ ........ 68 Chapter Three: Convergent Approach to Synthesis 2,5 Terpyrimidinylene Based Derivatives ................................ ................................ ................................ 70 3.1 Introduction ................................ ................................ ................................ ...... 70 3.1.1 Optimization of synthetic route to the 2,5 terpyrimidinylene scaffold ................................ ................................ .............................. 70 3.1.2 Modifications that m ight lead to improvement of the bioactivities ................................ ................................ ....................... 70 3.1.3 Problems existing in the developed synthetic process ...................... 73 3.1.4 Convergent synthetic strategy ................................ ........................... 74 3.2 Results and Discussion for the synthe sis of the new unsaturated ketones ................................ ................................ ................................ ............ 75 3.2.1 Retrosynthetic to make the unsaturated pyrimidineketones ................................ ................................ ......... 75 3.2.2 Attempted synthesis of the unsaturated pyrimidineketones 3.5 ................................ ................................ ... 76

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iii 3.2.3 Attempted synthesis of unsaturated ketones using 1,2,3 triazole ring ................................ ................................ .............. 77 3.2. 4 Biological testing of 3.10 ................................ ................................ .. 80 3.3 Conclusion ................................ ................................ ................................ ....... 80 3.4 Experimental Procedures ................................ ................................ ................. 80 3.5 References ................................ ................................ ................................ ........ 88 Chapter Four: Design and Synthesis of Hybrid Scaffold Non peptidic Helical Mimetics ................................ ................................ ................................ ..... 90 4.1 Introduction ................................ ................................ ................................ ...... 90 4.1.1 General scaffold of non peptidic helical mimetics ....................... 90 4.1.2 New library design strategy ................................ .............................. 91 4.1.3 Introduction of the hybrid scaffold ................................ ................... 91 4.1.4 Strategy to extend the pyrimidine rings using Huisgen Cycloaddition ................................ ................................ .................... 92 4.2 Results and Discussion for introducing 1,2,3 triazole to the scaffold ............. 93 4.2.1 Synthesis of 4 triazole pyrimidine hybrid dimer 4.6 ........................ 93 4.2. 2 Benefits of the 1,2,3 triazole ring ................................ ..................... 98 4.2.3 Synthesis of 2 triazole pyrimidin e hybrid dimer 4.13 ...................... 98 4.2. 4 Evaluation 1,2,3 triazole ring as a fragment in our scaffold ........... 101 4.3 Results and Discussion for introducing amino acids to the scaffold ............. 101 4.3.1 Possibility of introducing amino acids to the scaffold .................... 101 4.3.2 Testing of the new scaffold ................................ ............................. 102 4.3.3 Retrosynthesis of the trimer ................................ ............................ 103 4.3.4 Modification of the new target scaffold and the retrosynthesis route ................................ ................................ ......... 105 4.3.5 Synthesis of the trimer 4.2 1 ................................ ............................ 106 4.3.6 Properties of the timer 4. 2 1 ................................ ............................. 108 4.4 Biological testing of 4.6 and 4.13 ................................ ................................ .. 109 4.5 Conclusion ................................ ................................ ................................ ..... 110 4. 6 Experimental Procedures ................................ ................................ ............... 110 4.7 References ................................ ................................ ................................ ...... 128 Chapter Five : Design and Synthesis of Cyclic Urea HIV 1 Protease Inhibitor ............... 131 5 .1 Introduction ................................ ................................ ................................ .... 131 5 .1.1 AIDS ................................ ................................ ............................... 131 5 .1.2 Efforts to treat or cure AIDS ................................ ........................... 132 5.1.3 Structure of HIV ................................ ................................ ............. 132 5.1.4 HIV replication mechanism ................................ ............................ 134 5.1.5 Common targets to inhibit the HIV replication process ................. 135 5.1.6 Structure of HIV protease ................................ ............................... 136 5.1.7 HIV protease function mechanism ................................ .................. 137 5.1.8 Cyclic urea as t he HIV 1 protease inhibitor ................................ ... 137 5.1.9 Our design strategy ................................ ................................ ......... 138

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iv 5 .2 Results and Discussion ................................ ................................ .................. 139 5 .2.1 Retrosynthesis of 5 hydroxyl cyclic urea ................................ ....... 139 5 .2.2 Synthesis of intermediate 5.3 ................................ .......................... 140 5 .2.3 Photochemical electrocycl ization of 1,4,6 trisubstituted pyrimidin 2(1H) ones 5.1 ................................ ............................... 142 2. 3 Experimental Procedures of selected compounds ................................ .......... 1 47 2.4 References ................................ ................................ ................................ ...... 151 Appendix A: Selected 1 H and 13 C NMR Spectra ................................ ............................. 153 Appendix B: Selected HRMS ................................ ................................ .......................... 214 Appendix C: X Ray Crystallographic Data ................................ ................................ ..... 23 3 About the Author ................................ ................................ ................................ ... End Page

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v List of Tables Table 1.1 Typical disease pathways that involve the interaction of helix ............... 3 Table 2.1 Calculated logP values for terphenylene and terpyrimidinylene analogs ................................ ................................ ................................ ....... 39 Table 2.2 Library of the synthesized pyrimidine monomers 2.4 ............................... 42 Table 2.3 Analogs of the synthesized pyrimidine dimers ................................ .......... 46 Table 2.4 Analogs of the synthesized pyrimidine trimers ................................ ......... 47 Table 5.1 Analogs of the synthesized 1 ,4,6 trisubstituted pyrimidin 2(1H) ones 5.1 ................................ ................................ ................................ .... 141

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vi List of Figures Figure 1.1 Covalent bonding cyclic peptides ................................ ................................ 2 Figure 1.2 The smallest reported helical peptide in water and its conformation ................................ ................................ ................................ 6 Figure 1.3 Metal coordinate bonding cyclic peptides ................................ ................... 6 Figure 1.4 Cyclic peptide formed by unnatural amino acids ................................ ........ 7 Figure 1.5 Conformational change of cyclic peptide following the change of nitrogen nitrogen double bond conformation ................................ .............. 8 Figure 1.6 Short chain helix stabilized by mimiprotei n ................................ ............ 9 Figure 1.7 Examples of foldamers ................................ ................................ .............. 10 Figure 1.8 Comparison of helix and peptide helix ................................ ............... 11 Figure 1.9 First reported synthetic non peptidic helical mimetics .......................... 12 Figure 1.10 Model of a typical helix ................................ ................................ ......... 1 3 Figure 1.11 Model of the common helix binding residues ................................ ........ 1 4 Figure 1.12 Comparison of 1,4 terphenylene scaffold with an helix ........................ 1 5 Figure 1.13 Other helical mimetic scaffolds developed by Hamilton and coworkers ................................ ................................ ................................ ... 16 Figure 1.14 Helical mimetic scaffolds developed by Rebek and coworkers ............ 17 Figure 1.15 a) T he trisubstituted imidazole scaffolds ; b) one molecule from the scaffold docking into the binding site of the Bad/Bcl x L ..................... 1 8 Figure 1.16 Structure of chalcone C ................................ ................................ .............. 1 9

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vii Figure 1.17 (Left) t rans Fused polycylic ethers scaffold; (right) its superimposition with an helix ................................ ................................ 19 Figure 1.18 a) Structures of the nutlins; b) nutlin 2 superimposing with Phe 19 Trp 23 and Leu 26 of p53; c) nutlin 2 docking into the binding site of p53/MDM2 ................................ ................................ ............................ 2 1 Figure 1.19 a) Structures of 1,4 Benzodiazepine 2,5 diones; b) 1,4 Benzodiazepine 2,5 diones superimposing with Phe 19 Trp 23 and Leu 26 of p53 ................................ ................................ ............................... 22 Figure 1. 20 Structures of co activator binding inhibitors ................................ ............. 22 Figure 1. 21 Analogs of 1,4 terphenylene scaffold targeting different protein protein interactions ................................ ................................ ..................... 24 Figure 1. 22 p53 pathway to elicit apoptosis ................................ ................................ .. 28 Figure 1. 23 a) T he MDM2 binding cleft; b) c rystal structure of p53 binding into the MDM2 cleft ................................ ................................ .................. 29 Figure 1.24 Bak binding with the Bcl x L ................................ ................................ ...... 30 Figure 2.1 MDM2 an tagonist a and its docking in MDM2 ................................ ................................ ................................ ... 36 Figure 2.2 Recently reported non peptidic b k ................................ ................................ ........................ 38 Figure 2.3 Geometry of phenyl ring and pyrimidine ring ................................ ........... 40 Figure 2.4 Crystal structure of pyrimidine dimer ................................ ........................ 46 Figure 3. 1 terphenylene derivatives .............................. 70 Figure 3. 2 Synthesized 2,5 terpyrimidinylene derivatives ................................ .......... 71 Figure 3. 3 Structure comparison of 1,4 terphenylene and 2,5 pyrimidinylene scaffold ................................ ................................ ................................ ....... 71 Figure 4.1 Abstract structure of helical mimetics ................................ ................... 91 Figure 4.2 Trimer with an amino acid in the scaffold fitting the abstract scaffold ................................ ................................ ................................ ..... 102

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viii Figure 4.3 Possible conformation of the new scaffold ................................ .............. 103 Figure 4.4 Elisa results of 4.6 and 4.13 ................................ ................................ ..... 109 Figure 5.1 Estimated number of people living with HIV globally, 1990 2007 ........ 131 Figure 5.2 Diagram of HIV ................................ ................................ ....................... 133 Figure 5.3 HIV replication cycle ................................ ................................ ............... 135 Figure 5.4 Structure of HIV PR complexed with TL 3 ................................ ............. 136 Figure 5.5 Mechanism of HIV PR proteolysis ................................ .......................... 137 Figure 5.6 X ray crystal structure of cycl ic urea in the active site of HIV PR ......... 138 Figure 5.7 UV absorption of 5.1a ................................ ................................ .............. 144

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ix List of Schemes Scheme 2.1 Synthesis of pyrimidine rings ................................ ................................ .... 40 Scheme 2.2 Retrosynthesis of 1,4 terpyrimidinylene ................................ .................... 41 Scheme 2.3 Synthesis of the pyrimidine monomers 2.4 ................................ ................ 42 Scheme 2.4 Synthetic strategy to convert sterically hindered nitriles to amidines ....... 43 Scheme 2.5 Reaction and side reaction of hydroxylamine nucleophilic addition ......... 44 Scheme 2.6 Proposed transition state of hydroxylamine nucleophilic addition ............ 45 Schem e 2.7 Synthesis of the pyrimidine dimer ................................ ............................. 45 Scheme 2.8 Synthesis of the pyrimidine trimers ................................ ........................... 47 Scheme 3.1 Reaction and side reaction of hydroxylamine nucleophilic addition ......... 73 Scheme 3.2 Reaction and side reaction of amidine condensation reaction ................... 73 Scheme 3.3 Retrosynthesis using the convergent strategy ................................ ............ 74 Scheme 3.4 Retrosynthesis of the unsaturated pyrimidineketones ..................... 75 Scheme 3.5 Attempted synthesis of compound 3.5 ................................ ....................... 76 Scheme 3.6 Hydroxylamine addition of compound 3.2 ................................ ................ 77 Scheme 3.7 Synthes is of the unsaturated triazoleketones ................................ .. 78 Scheme 3.8 Condensation to synthesize the hybrid trimer 3.11 ................................ .... 79 Scheme 4.1 Synthetic strategy to make the hybrid dimer ................................ ............. 93 Scheme 4.2 Synthesis of hybrid dimer 4.6 ................................ ................................ .... 94

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x Scheme 4.3 Comparison of different substituted 1,3 diketones reacting with DMF DMA ................................ ................................ ................................ 95 Scheme 4.4 One possible mechanism of 1,3 diketone reacting with DMF DMA ........ 96 Scheme 4.5 General methodology to synthesize u nsaturated ketones from ketones ................................ ................................ ................................ ....... 99 Scheme 4.6 Synthesis of hybrid dimer 4 .13 ................................ ................................ 100 Scheme 4.7 Retrosynthesis of the hybrid trimer ................................ .......................... 104 Scheme 4.8 Synthesis of the 2 chloro pyrimidine ring ................................ ............... 105 Scheme 4.9 Modification of the target molecule ................................ ......................... 106 Scheme 4.10 Synthesis of trimer 4.22 ................................ ................................ ........... 107 Scheme 5.1 Retrosynthesis of 5 hydroxyl cyclic urea ................................ ................ 139 Scheme 5.2 Synthesis of the intermediate 5.3 ................................ ............................. 140 Scheme 5.3 Hydrolysis of N benzylure a under high temperature acidic conditions ................................ ................................ ................................ 141 Scheme 5.4 Synthesis of the 1,3 diazetidine 2 one intermediate ................................ 143 Scheme 5.5 Side prod uct of the oxidation of 5.2a ................................ ....................... 145 Scheme 5.6 Equilibrium between 5.3 and its water added form .......................... 14 6

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xi List of Abbreviations Alpha Angstrom Ac 2 O Acetic anhydride AcOH Acetic acid aq. Aqueous Beta BINAP 2,2' bis(diphenylphosphino) 1,1' binaphthyl Bn Benzyl Boc tert Butoxycarbonyl br Broad (spectral) Bu Butyl o C Degree Celsius 13 C NMR Carbon 13 Nuclear Magnetic Resonance CDI N,N' Carbonyldiimidazole CH 3 CN Acetonitrile Cs 2 CO 3 Cesium carbonate Delta or chemical shift DCM Dichloromethane DIEA Diisoprop ylethylamine DMF N,N Dimethylformamide DMF DMA N,N dimet hylformamide dimethyl acetal DMSO Dimethylsulfoxide ELISA Enzyme linked immunosorbent assay Et Ethyl EtOAc Ethyl acetate EtOH Ethanol ESI Electrospray ionization equiv. Equivalent(s) FP Fluorescence polarization g Gram(s) 1 H NMR Proton Nuclear Magnetic Resonance h Hour(s) HPLC High pressure liquid chromatography HR High resolution Hz Hertz IC 50 50% inhibitory concentration J Coupling constant(s)

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xii Ki Inhibitor dissociation constant KOBt P otassium tert butoxide KOt amyl P otassium tert amyloxide Leu Leucine LiOH Lithium hydroxide M Molar or moles per liter MDM2 Murine double minute 2 Me Methyl MeOH Methanol mg Milligram(s) min Minute(s) mL Milliliter(s) mmol Millimole(s) m.p. Melting point MS Mass spectrum MW Microwave NaH Sodium hydride NaOEt Sodium ethoxide NaOH Sodium hydroxide nM Nanomolar ORTEP Oak Ridge thermal ellipsoid plot (crystallography) PCC P yridinium chlorochromate Pd/C Palladium on carbon Ph Phenyl Phe Phenylalanine ppm Parts per million Pr Propyl RM S D Root mean square deviation rt Room temperature SAR Structure activity relationship sat. Saturated TFA Trifluoroacetic acid THF Tetrahydrofuran TMS Cl Trimethylsilyl chloride TLC Thin layer chromatography Trp Trptophan TS Tumor suppressor Microliter(s) Mic r omolar Val Valine wt Wild type

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xiii Design and Combinatorial Synthesis Approach of Non peptidic Trimeric Small Molecules Mimicking i i +4(3), i +7 Positions of Helices M ing zhou Zhou A bstract Protein protein interactions are key to several biological processes that facilitate signal transduction and many other processes. These interactions are involved in pathways that are critical to many human diseases. Targeting specific protein protein interactions is a challenging goal bec ause protein protein interactions are predominately through hydrophobic interactions. Antagonists of the protein protein interactions need to be perfectly fit into the binding pockets to ensure the activity. The helical domain of the proteins behaves as the recognition motifs for numerous protein protein, and protein nucleic acid interactions Research has shown that pathways of many diseases contain protein protein interactions involv ing helical domains, e.g. neurological disorders bacterial infec tions HIV and cancer, etc. It is difficult yet very important to design small molecules to target the shallow binding areas of protein protein interactions So far the 1,4 terphenylene scaffold which has been used to t arget the interactions between p53/MDM2, Bak/Bcl x L etc Inspired by this, we design ed and synthesized three new scaffolds of non pepditic helical mimetics, mimicking the i i + 4, i + 7 positions of an helix There are three basic principles that were leading our design. The side chains of our designed molecules should act as mimetics of the side chains of an helix. Second our molecules should possess

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xiv improved water solubility. Third, the molecules should be easy to synthesize to generate a f ocused library. Some of our molecules, including the one s whose molecular weight are as low as 294, started to show some inhibition against p53/MDM2 interaction s.

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1 Chapter One: Peptidic and Non-peptidic Scaffolds to Disrupt Protein-protein Interactions that Involve -Helices 1.1 Protein-protein interactions Protein-protein interactions are key to several biological processes that facilitate signal transduction and many other processes. The se interactions are involved in pathways that are critical to many human diseases. By disrupting certain protein-protein interactions, we are able to gain an understanding of functions of individual proteins and their roles in the biological system; most importantly, this allows us to gain an understanding of the role of specific proteins in disease pathways. 1.2 Efforts to target the protein-protein interaction Targeting specific protein-protein interactions is a challenging goal because unlike the interactions between the enzymes and their substrates which contain many hydrogen bonds, protein-protein interactions are predominately through hydrophobic interactions. Antagonists of the protein-protein interactions need to be perfectly fit into the binding pockets to ensure the activity. Unfortunately, the binding areas on the proteins are normally shallow and large (Stites, 1997), unlike that of the enzymes; this makes the design of small molecule antagonists that can disrupt protein-proteins interactions even more difficult (Ernst et al., 2003). Screening chemical libraries has not

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2 been particularly successful in this area (Kutzki et al., 2002). However, rational design has proven to be a more suitable approach (Yin and Hamilton, 2005). Rational design is a more successful procedure because the binding sites of the protein-protein interaction s are normally well-defined three-dimensional surfaces; hence, molecules with these properties have the most potential to serve as protein-protein interaction inhibitors. Protein-protein interactions occur on shallow surface features of the protein; typically, the binding areas contain only a limited amount of crucial amino acid residues. The most rational way to disrupt these interactions is to design and synthesize molecules that are capable of mimic king the three dimensional configuration of those crucial amino acids side chains. The two essential factors in designing small molecule protein-protein interaction antagonists are to identify the three dimensional configurations of the crucial binding amino acids of the target interactions and to design scaffolds that could hold the functional groups at specific locations. 1.3 Efforts to target the protein-protein interactions involving -helix One of the most important and abundant protein secondary structures is the helix. The -helical domain of the proteins behaves as the recognition motifs for numerous protein-protein interactions, protein-DNA interactions, and protein-RNA interactions (Lavery, 2005) ; (Klug, 2005). Research has shown that pathways of many diseases contain protein-protein interactions involve -helical domains (Table 1.1) (Davis et al., 2007).

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3 A typical -helix has a structural domain, which is about 15 25 residues in length (Garner and Harding, 2007). The -helix residue peptides when excised from their parent proteins can not retain their secondary structures (Kelso and Fairlie, 2003). In addition, the excised peptides, in vivo could not bind to their target protein any more. This is the reason why we cannot use the -helix sequence directly as the antagonist for protein-protein interactions. Table 1.1: Typical disease pathways that involve the interaction of -helix ppi diseases motif Tachykinin receptors/peptide neurological disorders i i + 4 NaIP bacterial infections i i + 4, i + 7 gp41 HIV i i + 3, i + 4, i + 7 p53/MDM2 cancer i i + 4, i + 7 smMLCK/CaM cancer i i + 4, i + 7 Rac1/Tiam1 cancer i i + 4, i + 7 GRIP1/Era cancer i i + 3, i + 4 Vav cancer i i + 1, i + 4 Bak/Bcl x L cancer i i + 4, i + 7, i + 11 Due to the importance of the -helix in the field of protein-protein interactions extensive research has focused on the design and synthesize of -helical mimetics. Studies of -helical mimetics are important because it can help us understand the roles that -helices play in protein-protein interactions. 1.4 Peptidic -Helical mimetics

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4 -Helices are the secondary structures of peptide chains; thus, it is straightforward to design their mimetics using peptides. As a matter of fact, peptidic helical mimetics are a successful approach toward mimicking -helical protein-protein interactions. 1.4.1 Cyclic peptides Despite the fact that many peptidic -helical mimetic scaffolds have been developed, the strategy is similar. As discussed above, short peptide chains are prone to remain unfolded, even though the same primary sequences form -helices in proteins. To make short stable peptide -helices, the peptides could be cyclized so that their conformational flexibility is locked. In other words, cyclization of peptides is the most common strategy that peptide chemists use to stabilize short chain peptides to form helices. The peptide cyclization strategy is a simple procedure. Because the side chains of the amino acid residues of an -helix at the i i + 4, i + 7, i + 11 positions are on the same face, if we connect two side chains among the i and the i + 4, i + 7, and i + 11 positions of an unfolded peptide, with proper length linker, to force them be on the same face, the peptide is stabilized as an -helical conformation. There are three major methodologies that have been developed following this strategy. 1.4.2 Direct covalent bonding of the side chains of the amino acids

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5 There are two kinds of covalent bonds that could be directly formed between the side chains of the natural amino acids: amide bonds between lysine and aspartic acid / glutamic acid (Condon et al., 2000), and disulfide bonds between two cysteines (Jackson et al., 1991) (Figure 1.1). Figure 1.1: Covalent bonding cyclic peptides The synthesis of these kinds of cyclic peptides is convenient because coupling of side chains of lysine and aspartic acid / glutamic acid forms an amide bond and the oxidation of two sulfides on cysteine side chains forms a disulfide bond. It has been reported that this direct covalent bonding methodology has been utilized to make the smallest -helical peptide in water (Figure 1.2) (Shepherd et al., 2005). However, this is also the limitation of using direct covalent bonding. The side chains of the amino acid have limited length; consequently, this method could not be used to make larger -helical regions. When a relatively longer unfolded -helical peptide chain has been cyclized using this method, only the cyclized small region could be an -helix (Garner and Harding, 2007).

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6 Figure 1.2: The smallest reported -helical peptide in water and its conformation determined by NMR 1.4.3 Metal coordinate bonding Besides the direct covalent bonding between the side chains of two amino acids, coordinate bond was used to connect the side chains (Figure 1.3) (Kelso et al., 2003) ; (Kelso et al., 2004) ; (Beyer et al., 2004). Figure 1.3: Metal coordinate bonding cyclic peptides Transition metals, such as palladium, zinc, etc. were used to form the coordinated bonds with side chains of histidine / cysteine at i i + 4 positions (Figure 1.3).

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7 Like the direct covalent bonding, only small -helical regions could be formed by metal coordinate bonding. Also, these cyclic peptides are not stable in water. However, it provides one other possible way to form an -helix; especially since metal ions play important roles in biological systems. 1.4.4 Connecting side chains of unnatural amino acids at i and i + 7 positions. All natural amino acids have side chains which are short in length. This limits the potential size of the cyclic peptide if we choose to directly connect the side chains. There are two ways to solve this problem; one is to use unnatural amino acids with longer side chains, or longer linkers can be used. For the first way, unnatural amino acids were used, which have extended sid e chains (5 to 8 carbons-carbon single bond s in length) introduced at the i and i + 7 positions (Figure 1.4) (Blackwell and Grubbs, 1998) ; (Blackwell et al., 2001). Figure 1.4: Cyclic peptide formed by unnatural amino acids Carbon-carbon double bonds were used to connect the side chains. Although stable in water, amide bonds and disulfide bonds are ruled out because they are prone to

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8 be degraded in biological systems. In summary, two amino acids that have side chains with an alkene group at the terminal position are introduced to the peptide scaffold through the Grubbs metathesis coupling reaction. 1.4.5 Connecting i and i + 7 side chains with a photo-controlled azobenzene linker The second strategy to form a bigger -helical region is to use longer linkers to connect the i and i + 7 positions. Azobenzene was chosen as this linker because of its length and its photo sensitive isomerization (Woolley, 2005) ; (Ihalainen et al., 2007). The azobenzene linker was connected to the peptide by the help of a sulfur atom at the side chains of the cysteine. One unique property of azobenzene is that its nitrogennitrogen double bond isomerizes under different conditions (Figure 1.5) ; the distance of the benzene rings changes greatly between cis and trans double bonds. The azobenzene linker was designed so that, when the nitrogen-nitrogen double bond is in the cis configuration, the peptide will be stabilized as an -helix. Figure 1.5: Conformational change of cyclic peptide following the change of nitrogennitrogen double bond conformation

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9 However, when the double bond is in trans configuration, the peptide chains would be unable to maintain the -helical secondary structure. The azobenzene linker strategy is one method that can control the conformation of a peptide chain after the peptide has been cyclized; thus could be applied to understand the roles that -helices play. 1.5 Miniproteins Short peptide sequences taken from a protein tend to lose their secondary structures. This also suggests that although the other part of the protein does not participate in the bonding interaction, it helps stabilize the protein secondary structure. I f a second protein was in troduced to stabilize the short peptide chain, will the peptide reform its secondary structure? Figure 1.6: Short chain -helix stabilized by mimiprotein

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10 Following this question, miniproteins were designed to stabilize the non-bonding face of a -helix through protein-protein interactions, so that a short peptide chain will form its secondary structure (Figure 1.6) (Chin and Schepartz, 2001) ; (Rutledge et al., 2003). Miniproteins are small proteins that have well defined secondary structures. They can recognize and interact with short peptide chains, forcing the peptide chains to form secondary structures, normally an -helix. The now well-assembled short peptides behave like the -helical domains in the proteins to recognize their targets. 1.6 Foldamers Figure 1.7: Examples of foldamers

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11 Foldamers are oligomers have well-defined secondary structures stabilized by non-covalent bonding. The peptidic foldamers use unnatural peptidic building blocks, such as -peptides, -peptides, -peptides, peptoid, and oligourea, etc. (Figure 1.7) (Werder et al., 1999) ; (Cheng, 2004) ; (Kritzer Joshua et al., 2005). Foldamers not only have secondary structures similar to -helix (Figure 1.8), but are also capable of disrupting protein-protein interactions involving -helices. Figure 1.8: Comparison of -helix and -peptide helix 1.7 Synthetic non-peptidic -helical mimetics with linear scaffold Besides the peptidic approach to mimic the -helices, organic chemists have built small molecular (<750 Da) scaffolds in this field. Until now, most drugs on the market are small synthetic molecules. Small molecules have several advantages to serve as the drug molecules, such as being more stable and more membrane permeable. It is difficult for small molecules to target the sh allow binding areas of proteinprotein interactions; it is even more difficult for them to mimic the complicated surface

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12 areas of -helices. This might be the reason why the first synthetic non-peptidic helical mimetic small molecules (Figure 1.9) were not reported until 2001 (Garner and Harding, 2007). Figure 1.9: First reported synthetic non-peptidic -helical mimetics Hamilton and coworkers were the first to publish synthetic small molecular nonpeptidic -helical mimetic scaffold, 1,4-terphenylene (Orner et al., 2001). Although the surface of an -helix is complicated, its binding site is along one face of the -helix Therefore, we just need to design molecules to mimic that face of the -helix. Also, an -helix has a coil-like backbone; and its configuration is rigid, regardless of the amino acid residues. This means that only residues lined in certain primary structures sequence (e.g. i i + 3, i + 4; i i + 3, i + 7; i i + 4, i + 7; i i + 4, i + 7, i + 11 ) (Figure 1.10 ) could be on the same face with each other. Thus, the amino acid residues on one face of the helix should have fixed relative geometric locations to each other.

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13 Figure 1.10: Model of a typical -helix (NIH) The relative location between the amino acid residues on an -helix can be measured by two factors, the translation along the helical axis, which is 1.5 per two re sidues and the turn angle, which is 100 o per residue (wiki). Figure 1.11 shows the relative location of the i i + 3, i + 4; i i + 3, i + 7; and i i + 4, i + 7, when seen from both the helical axis (a and b) and the side of the -helix (c). Part a) showed the relative location of the residues when seen from the helical axis. As we can see that the i i + 3, i + 4; i i + 3, i + 7; and i i + 4, i + 7 residues are all on one face of the -helix. Part b) eliminated the -helix backbones, an d shows only the relative location among the residues when seen from the helical axis. Part c) added the second factor into consideration translation along the helical axis, showing their relative location when seen from the side of the -helix.

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14 Figure 1.11: Model of the common -helix binding residues 1.7.1 -Helical mimetic scaffolds developed by Hamilton and coworkers

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15 -terphenylene scaffold came from the fact that the distance between its side chains of the phenyl rings matches that of the i i + 4, i + 7 (Figure 1.12); while the slightly twist ed 1,4-terphenylene scaffold pointed its side chains to similar side chain orientations of the -helix residues. Figure 1.12: Comparison of 1,4-terphenylene scaffold with an -helix The discovery of the 1,4-terphenylene scaffold has removed a lot of successive work. Figure 1.13 shows the newer scaffolds reported by Hamilton and coworkers (Ernst et al., 2003) ; (Davis et al., 2005); (Estroff et al., 2004) ; (Yin et al., 2005b) ; (Rodriguez and Hamilton, 2006) ; (Kim and Hamilton, 2006). However, none of the newer scaffold are as potent as the original 1,4-terphenylene one.

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16 Figure 1.13: Other -helical mimetic scaffolds developed by Hamilton and coworkers 1.7.2 -Helical mimetic scaffolds developed by Rebek and coworkers The 1,4-terphenylene series of non-peptidic -helical mimetics has not only inspired the Hamilton group, but also inspired chemists all over the world. Figure 1.14 showed the scaffolds developed by Rebek and coworkers (Volonterio et al., 2007) ; (Moisan et al., 2008).

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17 Figure 1.14: -Helical mimetic scaffolds developed by Rebek and coworkers 1.7.3 Trisubstituted imidazole scaffold One disadvantage of the 1,4-terphenylene series is that it is very challenging to synthesi ze Antuch and co-workers at tempted to solve this problem by modifying the 1,4-terphenylene scaffold to a trisubstituted imidazole (Figure 1.15), which could easily be synthesized by multicomponent reactions (Antuch et al., 2006).

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18 Figure 1.15: a) the trisubstituted imidazole scaffolds; b) one molecule from the scaffold docking into the binding site of the Bad/Bcl-x L 1.7.4 Benzylideneacetophenone scaffold This scaffold is based on chalcones, which have some intrinsic antitumor activities. Benzylideneacetophenones are planar molecules that mimic the i i + 4, i + 7 positions of -helices (Stoll et al., 2001). Figure 1.16 shows the structure of chalcone C and the way it mimics the -helix.

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19 Figure 1.16: Structure of chalcone C 1.7.5 trans -Fused polycyclic ethers scaffold Polycyclic ethers are conceptually inspired by the skeletal feature of marine toxins. The distance between R 1 and R 2 group (4.8 ) is very close to that between i i + 4 residues (5.4 ) (Figure 1.17) (Oguri et al., 2005). Figure 1.17: (Left) trans-Fused polycylic ethers scaffold; (right) its superimposition with an -helix

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20 1.8 Synthetic non-peptidic -helical mimetics with other scaffold We can consider -helices as a linear backbone. There are several reported potent non-peptidic -helical mimetics that do not have a linear backbone. An example of this type of mimetic is Nutlin which is the most potent p53/MDM2 antagonist thus far reported. The problem is that researchers have not developed a general strategy to design th ese kinds of -helical mimetic scaffolds. 1.8.1 Nutlins Nutlins were discovered while screening a diverse library of synthetic chemicals (Vassilev et al., 2004), of which, nutlin-2 demonstrated IC 50 = 140 nM against p53/MDM2 interactions. Nutlins represent a design strategy that is different from Hami in which all have linear backbones that mimic the coil of an helix. Three of the four substitutions on the imidazoline ring mimic the i i + 4, i + 7 position of the p53 N-terminal -helix. (Figure 1.18)

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21 Figure 1.18: a) Structures of the nutlins; b) nutlin-2 superimposing with Phe 19 Trp 23 and Leu 26 of p53; c) nutlin-2 docking into the binding site of p53/MDM2 1.8.2 1,4-Benzodiazepine-2,5-dione scaffolds 1,4-Benzodiazepine-2,5-diones (Figure 1.19) was also found as a hit when screening a library (Cummings et al., 2006). 1,4-Benzodiazepine-2,5-diones have a heterocyclic backbone, which is similar to that of the nutlin molecules.

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22 Figure 1.19: a) Structures of 1,4-Benzodiazepine-2,5-diones; b) 1,4-Benzodiazepine-2,5diones superimposing with Phe 19 Trp 23 and Leu 26 of p53 1.8.3 Co -activator binding inhibitors Figure 1.20: Structures of co-activator binding inhibitors

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23 Co -activator binding inhibitors also contain a heterocyclic backbone (Figure 1.20); they mimic the i i + 3, i + 4 positions of an -helix (Antuch et al., 2006). 1.9 Summary of non-peptidic -helical mimetics We have summarized the reported non-peptidic scaffolds that could mimic the helices. Both scaffolds with linear backbones and heterocycl ic backbones have proven to be potential -helical mimetics. The idea is that, the backbones should be capable to assist the hydrophobic side chains to be positioned at the right three-dimensional location. At present, the linear backbone design is more popular because this design also mimics the backbone coil of -helix making it more straightforward. Those molecules showed potential for synthetic scaffolds to mimic the configuration of three or four critical amino acid residues on a -helix. The challenge and the joy in designing non-peptidic -helical mimetics are to understand the three dimensional structures of the molecules. 1.10 Possibility of designing general non-peptidic -helical scaffolds Most of the reported non-peptidic -helical mimetic molecules were design ed to target one specific protein-protein interaction, such interactions between p53/MDM2, Bak/Bcl-x L etc. However, as analyzed before (1.3.5), the binding configuration should be the same, regardless of the amino acid residues. So a question emerged: is it possible to develop a general non-peptidic -helical scaffold?

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24 Figure 1.21: Analogs of 1,4-terphenylene scaffold targeting different protein-protein interactions

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25 This is how nature accomplishes it, as least. NaIP, p53/MDM2, smMLCK/CaM, and Rac1/Tiam1 protein-protein interactions all share the i i + 4, i + 7 binding configuration (Table 1.1). As a matter of fact, the 1,4-terphenylene backbones have been used to target several different protein-protein interactions. By replacing the side chains of the 1,4-terphenylene scaffold, the analogs could be used to target different proteinprotein interactions (Figure 1.21) (Orner et al., 2001) ; (Ernst et al., 2002) ; (Kutzki et al., 2002) ; (Yin et al., 2005a). This shares the same logic that nature adapted to interact with different protein-protein interactions by changing the amino acids residues on the helices. Although sharing one scaffold, the 1,4-terphenylene scaffold molecules also showed selectivities between the different protein-protein interactions. For example, Hamilton and co-workers stated that the 1,4-terphenylene scaffold demonstrated selectivity between p53/MDM2 and and Bcl-x L by switching between one naphthyl group (Davis et al., 2007). 1.11 Balance between rational design and combinatorial chemistry Due to the ease in controlling the well-defined three dimensional surface configurations of the mimetics, rational design has proven to be a more effective way to prepare -helical mimetic scaffolds. One difficulty associated with most of the structurally well-defined scaffolds developed so far is they are difficult to synthesize. This has become a major factor that delays chemists from developing more potent -helical mimetics.

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26 The strategy used in our research was to modify our target scaffolds so that they are suitable for combinatorial synthesis. As we were focusing on the rational design and synthesis of scaffolds with linear backbones, we divided the backbone in to several modules, each of which was corresponding to one of the i i + 4, i + 7 position side chains. In summary, we tried to find a balance between the well-defined configuration and ease of synthesis by refining our target molecules. 1.12 Cancer: our target Our goal was to develop -helical mimetic scaffolds that were easy to build, so that we could synthesize focus ed libraries rapidly. The se libraries should be general for all protein-protein interactions that involve -helices. However, our group in the Moffitt Cancer Center, is interested in their anti-cancer properties, specifically, their effects in apoptosis. Cancer has been identified as a genetic disease; it is a result of a combination of deregulated cell proliferation and suppressed apoptosis (Green and Evan, 2002). 1.12.1 Hallmarks of cancer There are six factors that identify cancer cells from normal diseases (Hanahan and Weinberg, 2000). First, there is self-sufficiency in the growth signal. Healthy cel ls need extracellular growth signals, while cancer cells are capable of generating their own growth signals. Second, cancer cells are insensitive to anti-growth signals. Antigrowth

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27 signals in normal cells can temporarily or permanently deactivate the proliferation. Cancer cells block the antigrowth signals pathways, thus are inert to the signals. Third, cancer cells evade apoptosis. Apoptosis is defined as programmed cell death. When DNA damage is sensed, apoptosis will be elicited to kill the damaged cell. Fourth, there is a l imitless replicative potential. Normal cells have a finite replicative potential (60-70 doublings for normal human cell types), while cancer cells do not. The fifth factor that identifies cancer cells is that there is a sustained angiogenesis. Angiogenesis is the growth of new blood vessels which are used to supply oxygen and nutrients. This process stops once a tissue is formed. Cancer cells manage to maintain the constant growth of blood vessels so that they can continue to grow. Finally, cancer cells have the capability to invade tissue and metastasize. The primary tumor mass releases cancer cells to colonize new areas in other parts of the body. This is the cause of about 90% of human cancer death. 1.12.2 Apoptosis As stated before, apoptosis is programmed cell death. Multicellular animals re gularly need to remove ex cess cells and eliminate damaged cells. Avoiding apoptosis is essential for the survival of cancer cells. Apoptosis can be divided into be two process es sensors and effectors (Hengartner, 2000). Pro-apoptosis is initiated by the release of cytochrome C, which is regulated by the Bcl-2 family of proteins. In turn, the Bcl-2 family is, regulated by p53, which is the tumor suppressor protein.

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28 When DNA damage is sensed, p53 will be released to upregulate the expression of Bax. Bax is a proapoptotic protein in Bcl-2 family; which elicits the release of cytochrome C (Figure 1.22) (SigmaAldrich). Th is process is called the sensor process. The released cytochrome C will then activate caspases -8 and/or -9, which are the enzymes that perform an array of proteolytic processes to kill the cell. This is the effector process. Figure 1.22: p53 pathway to elicit apoptosis 1.12.3 p53/MDM2 interaction p53 is a tumor suppressor protein, which helps maintain the integrity of DNA by eliciting apoptosis when DNA damage is detected. It has been found that the p53 pathway is lost in most human cancers. There are many ways that cancer cells evade apoptosis. The most common way is through the mutation of p53, which was detected in

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29 more than 50% of human cancers (Green and Evan, 2002). For the remaining cases p53 is detected bound to MDM2, causing it to stay in its inactive form. p53 is regulated by MDM2 by binding to it; but when DNA damage is detected, p53 should be released to initiate apoptosis. Cancer cells prevent the release of p53 by amplifying the expression of MDM2 gene, thus producing more of the MDM2 protein. I f the extra MDM2 could be inhibited, the cancer cells with wild-type p53 should be killed. The MDM2 protein is a small globular protein that has a small hydrophobic cleft at one face (Figure 1.23 a) (Kussie et al., 1996). This cleft is about 25 long, 10 at its widest place, 10 at its deepest location. It is formed by four helices constituting the bottom and the two sides and has two clusters of three-stranded -sheets closing the ends. Figure 1.23: a) the MDM2 binding cleft; b) crystal structure of p53 binding into the MDM2 cleft The p53 binding site is an -helix domain, formed from residues 18 to 26, of which Phe 19 ( i ), Trp 23 ( i + 4), and Leu 26 ( i + 7) are the key binding residues (Figure 1.23

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30 b ). This -helix domain is insert ed deeply into the hydrophobic cleft of MDM2, forming very strong van der Waals interaction with the hydrophobic residues of MDM2. 1.12.4 Bcl-2 family protein-protein interactions The Bcl-2 protein family plays a central role in regulating apoptosis. The family contains both pro-apoptotic (Bax, Bak, Bid, Bim) and anti-apoptotic (Bcl-2, Bcl-x L BclW) function al proteins. Homoand heterodimerization within the proteins are the key to modulate their functions; when pro-apoptotic proteins are released from the dimers, apoptosis will be promoted. Figure 1.24: Bak binding with the Bcl-x L The Bcl2 and Bcl-x L proteins contain four Bcl-2 homology (BH) domains (BH1BH4), as well as a C-terminal hydrophobic tail. Bax and Bak contain almost the same components of Bcl2 and Bcl-x L except for the BH4 domain. Bid and Bik only contain the BH3 domain. Among the BH1-BH4 domains, the BH3 domain is involved in

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31 eliciting the apoptosis process; all Bak, Bax, Bid and Bim share similar BH3 domains (Green and Evan, 2002). The binding site between Bcl-x L and Bak were studied by NMR experiments (Sattler et al., 1997). The binding site of Bak is an -helix domain (Figure 1.24) formed from residues 72 to 87, of which Val 74 ( i ), Leu 78 ( i + 4), Ile 81 ( i + 7) and Ile 85 ( i + 11) are the binding residues. This -helix binds to the hydrophobic domain on the surface of the Bcl-x L formed by the BH1, BH2, and BH3 domain. 1.13 References Antuch, W., Menon, S., Chen, Q.-Z., Lu, Y., Sakamuri, S., Beck, B., SchauerVukasinovic, V., Agarwal, S., Hess, S., and Doemling, A. (2006). Design and modular parallel synthesis of a MCR derived alpha -helix mimetic protein-protein interaction inhibitor scaffold. Bioorganic & Medicinal Chemistry Letters 16 1740-1743. Beyer, R. L., Hoang, H. N., Appleton, T. G., and Fairlie, D. P. (2004). Metal Clips Induce Folding of a Short Unstructured Peptide into an alpha -Helix via Turn Conformations in Water. Kinetic versus Thermodynamic Products. Journal of the American Chemical Society 126, 15096-15105. Blackwell, H. E., and Grubbs, R. H. (1998). Highly efficient synthesis of covalently cross-linked peptide helices by ring-closing metathesis. Angewandte Chemie, International Edition 37, 3281-3284. Blackwell, H. E., Sadowsky, J. D., Howard, R. J., Sampson, J. N., Chao, J. A., Steinmetz, W. E., O'Leary, D. J., and Grubbs, R. H. (2001). Ring-Closing Metathesis of Olefinic Peptides: Design, Synthesis, and Structural Characterization of Macrocyclic Helical Peptides. Journal of Organic Chemistry 66, 5291-5302. Cheng, R. P. (2004). Beyond de novo protein design de novo design of non-natural folded oligomers. Current Opinion in Structural Biology 14, 512-520. Chin, J. W., and Schepartz, A. (2001). Design and evolution of a miniature Bcl-2 binding protein. Angewandte Chemie, International Edition 40, 3806-3809.

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32 Condon, S. M., Morize, I., Darnbrough, S., Burns, C. J., Miller, B. E., Uhl, J., Burke, K., Jariwala, N., Locke, K., Krolikowski, P. H. et al. (2000). The Bioactive Conformation of Human Parathyroid Hormone. Structural Evidence for the Extended Helix Postulate. Journal of the American Chemical Society 122, 3007-3014. Cummings, M. D., Schubert, C., Parks, D. J., Calvo, R. R., LaFrance, L. V., Lattanze, J., Milkiewicz, K. L., and Lu, T. (2006). Substituted 1,4-benzodiazepine-2,5-diones as alpha -helix mimetic antagonists of the HDM2-p53 protein-protein interaction. Chemical Biology & Drug Design 67, 201-205. Davis, J. M., Truong, A., and Hamilton, A. D. (2005). Synthesis of a 2,3';6',3''Terpyridine Scaffold as an alpha -Helix Mimetic. Organic Letters 7, 5405-5408. Davis, J. M., Tsou, L. K., and Hamilton, A. D. (2007). Synthetic non-peptide mimetics of alpha -helices. Chemical Society Reviews 36, 326-334. Ernst, J. T., Becerril, J., Park, H. S., Yin, H., and Hamilton, A. D. (2003). Design and application of an alpha -helix-mimetic scaffold based on an oligoamide-foldamer strategy: Antagonism of the Bak BH3/Bcl-xL complex. Angewandte Chemie, International Edition 42, 535-539. Ernst, J. T., Kutzki, O., Debnath, A. K., Jiang, S., Lu, H., and Hamilton, A. D. (2002). Design of a protein surface antagonist based on alpha -helix mimicry: inhibition of gp41 assembly and viral fusion. Angewandte Chemie, International Edition 41, 278-281. Estroff, L. A., Incarvito, C. D., and Hamilton, A. D. (2004). Design of a Synthetic Foldamer that Modifies the Growth of Calcite Crystals. Journal of the American Chemical Society 126, 2-3. Garner, J., and Harding, M. M. (2007). Design and synthesis of alpha -helical peptides and mimetics. Organic & Biomolecular Chemistry 5, 3577-3585. Green, D. R., and Evan, G. I. (2002). A matter of life and death. Cancer Cell 1, 19-30. Hanahan, D., and Weinberg, R. A. (2000). The hallmarks of cancer. Cell (Cambridge, Massachusetts) 100, 57-70. Hengartner, M. O. (2000). The biochemistry of apoptosis. Nature (London) 407, 770-776. Ihalainen, J. A., Bredenbeck, J., Pfister, R., Helbing, J., Chi, L., van Stokkum, I. H. M., Woolley, G. A., and Hamm, P. (2007). Folding and unfolding of a photoswitchable peptide from picoseconds to microseconds. Proceedings of the National Academy of Sciences of the United States of America 104, 5383-5388.

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33 Jackson, D. Y., King, D. S., Chmielewski, J., Singh, S., and Schultz, P. G. (1991). General approach to the synthesis of short alpha -helical peptides. Journal of the American Chemical Society 113, 9391-9392. Kelso, M. J., Beyer, R. L., Hoang, H. N., Lakdawala, A. S., Snyder, J. P., Oliver, W. V., Robertson, T. A., Appleton, T. G., and Fairlie, D. P. (2004). alpha -Turn Mimetics: Short Peptide alpha -Helices Composed of Cyclic Metallopentapeptide Modules. Journal of the American Chemical Society 126, 4828-4842. Kelso, M. J., and Fairlie, D. P. (2003). Current approaches to peptidomimetics. Molecular Pathomechanisms and New Trends in Drug Research, 579-598. Kelso, M. J., Hoang, H. N., Oliver, W., Sokolenko, N., March, D. R., Appleton, T. G., and Fairlie, D. P. (2003). A cyclic metallopeptide induces alpha helicity in short peptide fragments of thermolysin. Angewandte Chemie, International Edition 42, 421-424. Kim, I. C., and Hamilton, A. D. (2006). Diphenylindane-based proteomimetics reproduce the projection of the i, i+3, i+4, and i+7 residues on an alpha -helix. Organic Letters 8, 1751-1754. Klug, A. (2005). Towards therapeutic applications of engineered zinc finger proteins. FEBS Letters 579, 892-894. Kritzer Joshua, A., Stephens Olen, M., Guarracino Danielle, A., Reznik Samuel, K., and Schepartz, A. (2005). beta-Peptides as inhibitors of protein-protein interactions. Bioorg Med Chem 13 11-16. Kussie, P. H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A. J., and Pavletich, N. P. (1996). Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science (Washington, D C) 274, 948-953. Kutzki, O., Park, H. S., Ernst, J. T., Orner, B. P., Yin, H., and Hamilton, A. D. (2002). Development of a potent Bcl-xL antagonist based on alpha -helix mimicry. Journal of the American Chemical Society 124, 11838-11839. Lavery, R. (2005). Recognizing DNA. Quarterly Reviews of Biophysics 38, 339-344. Moisan, L., Odermatt, S., Gombosuren, N., Carella, A., and Rebek, J., Jr. (2008). Synthesis of an oxazole-pyrrole-piperazine scaffold as an alpha -helix mimetic. European Journal of Organic Chemistry, 1673-1676. NIH http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mcb/ch3f6.gif

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34 Oguri, H., Oomura, A., Tanabe, S., and Hirama, M. (2005). Design and synthesis of a trans-fused polycyclic ether skeleton as an alpha -helix mimetic scaffold. Tetrahedron Letters 46, 2179-2183. Orner, B. P., Ernst, J. T., and Hamilton, A. D. (2001). Toward Proteomimetics: Terphenyl Derivatives as Structural and Functional Mimics of Extended Regions of an alpha -Helix. Journal of the American Chemical Society 123, 5382-5383. Rodriguez, J. M., and Hamilton, A. D. (2006). Intramolecular hydrogen bonding allows simple enaminones to structurally mimic the i, i +4, and i +7 residues of an alpha -helix. Tetrahedron Letters 47, 7443-7446. Rutledge, S. E., Volkman, H. M., and Schepartz, A. (2003). Molecular recognition of protein surfaces: High affinity ligands for the CBP KIX domain. Journal of the American Chemical Society 125, 14336-14347. Sattler, M., Liang, H., Nettesheim, D., Meadows, R. P., Harlan, J. E., Eberstadt, M., Yoon, H. S., Shuker, S. B., Chang, B. S., Minn, A. J. et al. (1997). Structure of BclxL Bak peptide complex: recognition between regulators of apoptosis. Science (Washington, D C) 275, 983-986. Shepherd, N. E., Hoang, H. N., Abbenante, G., and Fairlie, D. P. (2005). Single Turn Peptide Alpha Helices with Exceptional Stability in Water. Journal of the American Chemical Society 127, 2974-2983. SigmaAldrich http://www.sigmaaldrich.com/life-science/cell-biology/learningcenter/pathway-slides-and/atmp53-signaling-pathway.html Stites, W. E. (1997). Protein-Protein Interactions: Interface Structure, Binding Thermodynamics, and Mutational Analysis. Chemical Reviews (Washington, D C) 97, 1233-1250. Stoll, R., Renner, C., Hansen, S., Palme, S., Klein, C., Belling, A., Zeslawski, W., Kamionka, M., Rehm, T., Muehlhahn, P. et al. (2001). Chalcone Derivatives Antagonize Interactions between the Human Oncoprotein MDM2 and p53. Biochemistry 40, 336344. Vassilev, L. T., Vu, B. T., Graves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Kong, N., Kammlott, U., Lukacs, C., Klein, C. et al. (2004). In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2. Science (Washington, DC, United States) 303, 844-848. Volonterio, A., Moisan, L., and Rebek, J., Jr. (2007). Synthesis of pyridazine-based scaffolds as alpha -helix mimetics. Organic Letters 9, 3733-3736.

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35 Werder, M., Hauser, H., Abele, S., and Seebach, D. (1999). beta -Peptides as inhibitors of small-intestinal cholesterol and fat absorption. Helvetica Chimica Acta 82, 1774-1783. wiki http://en.wikipedia.org/wiki/Alpha-helix In. Woolley, G. A. (2005). Photocontrolling Peptide alpha Helices. Accounts of Chemical Research 38, 486-493. Yin, H., and Hamilton, A. D. (2005). Strategies for targeting protein-protein interactions with synthetic agents. Angewandte Chemie, International Edition 44, 4130-4163. Yin, H., Lee, G.-i., Park, H. S., Payne, G. A., Rodriguez, J. M., Sebti, S. M., and Hamilton, A. D. (2005a). Terphenyl-based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition 44, 2704-2707. Yin, H., Lee, G.-i., Sedey, K. A., Kutzki, O., Park, H. S., Orner, B. P., Ernst, J. T., Wang, H. -G., Sebti, S. M., and Hamilton, A. D. (2005b). Terphenyl-Based Bak BH3 alpha Helical Proteomimetics as Low-Molecular-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society 127, 10191-10196.

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36 Chapter Two: Design and Synthesis of 2,5-Terpyrimidinylenes as More Drug-Like 1,4-Terphenylene Mimeti cs 2.1 Introduction 2.1.1 -Terphenylene Scaffold and Related Work -terphenylene library produced an inhibitor that displays favorable in vitro activity towards p53/MDM2 heterodimerization (Figure 2.1, a)(Yin et al., 2005a). Figure 2.1: -MDM2 antagonist a and its docking in MDM2

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37 The 4-, and positions of this terphenylene scaffold are design ed to mimic the i i+4, and i+7 -helix. The series of molecules bind to the same sites that p53 binds to MDM2; while the side chains occupy the sites that the triad of p53 (F19, W23, and L26) uses to interact with MDM2. However, the design has one drawback which prevents it from being a drug candidate. Both the terphenyl scaffold and the 4position side chains are hydrophobic; thus the lipophilicity of this series of molecules is very high (Yin et al., 2005b). This is why the 1,4-terphenylene molecules have good bio-activities in fluorescence polarization ( FP ) assay, in which the 1,4-terphenylene molecule competitively replaced the p53 (residues 15 31) in interaction with MDM2 in vitro but not in in vivo assay (Yin et al., 2005a). Further investigation on this scaffold wa s carried out by the Hamilton group (Ernst et al., 2003), (Davis et al., 2005), (Estroff et al., 2004), (Yin et al., 2005b), (Rodriguez and Hamilton, 2006), as well as the (Moisan et al., 2008), (Volonterio et al., 2007) (Figure 2.2), but they were not as successful as the original 1,4-terphenylene a (Figure 2.1). Some were designed to mimic the interaction between Bcl-x L /Bak, but that interaction is also mediated through an helix, therefore the scaffold should show some similar properties.

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38 Figure 2.2: Recently reported non-peptidic -helical mimeti cs g k

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39 2.1.2 Design of the 2,5-terpyrimidinylene scaffold Since none of the newer -helical mimetics are as good as the original 1,4terphenylene ones, we hypothesized that the terphenyl scaffold might be the best antagonist for the p53/MDM interaction. Our group also found that by simply replacing the phenyl rings of the scaffold with pyrimidine rings, the calculated logP value drops by an average of 4 (Table 2.1), meaning that the corresponding terpyrimidinylene molecules are expected to be more water soluble and therefore be more bio-available. Table 2.1: Calculated logP values for terphenylene and terpyrimidinylene analogs R 0 CN CN CN CN R 0 NH 2 P h P h P h R 1 i Bu i Bu i Bu Bn R 2 Bn Bn CH 2 (1 Naph) CH 2 (1 Naph) R 3 i Bu i Bu i Bu i Bu logP(carbon) 8.6 10.4 11.3 12.2 logP(nitrogen) 4.7 5.8 7 7.8 logP(difference) 3.9 4.6 4.3 4.4 Furthermore, due to the geometrical similarity of phenyl and pyrimidine units (Figure 2.3 ), we reasoned that the 2,5-terpyrimidinylene series would have similar bioactivity, to the corresponding 1,4-terphenylene scaffold.

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40 Figure 2.3: Geometry of phenyl ring and pyrimidine ring 2.2 Results and Discussion for the 1,4-pyrimidinylene scaffold 2.2.1 Retrosynthesis of the 1,4-pyrimidinylene scaffold Our group developed a convenient methodology to synthesize pyrimidine rings with varying side chains (Scheme 2.1). We condensed commercially available formamidine/amidine/guanidine m with readily synthesized -unsaturated cyanoketones l at elevated temperatures in the presence of base, making a library of pyrimidines n with variable 2 and 4 positions. Scheme 2.1 Synthesis of pyrimidine rings The -unsaturated -cyanoketones l contains an -cyano group because this cyano group can potentially be converted in to the amidine group after the synthesis of the

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41 monomer n The newly generated amidine can react with another molecule of unsaturated -cyanoketone l to form a dimer p By repeating this process, we planned to synthesize the trimer o. The corresponding retrosynthetic strategy is shown in Scheme 2.2. Scheme 2.2: Retrosynthesis of 1,4-terpyrimidinylene 2.2.2 Synthesis of pyrimidine monom er 2.4 library The synthesis of pyrimidine monomers 2.4a g is shown in Scheme 2.3. Acetonitrile was deprotonated by potassium tert -pentoxide, and then reacted with various esters to produce -cyanoketones 2.2 (Ji et al., 2006), which then reacted with N,N dimethylformamide dimethyl acetal (DMF-DMA) to form the -unsaturated cyanoketones 2.3 (Reuman et al., 2008). Most of the ester starting materials are commercially available; those that are not can be easily prepared from the corresponding acid by Fisher esterification, using methanol as the solvent, and concentrated sulfuric acid

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42 Scheme 2.3: Synthesis of the pyrimidine monomers 2.4 Table 2.2: Library of the synthesized pyrimidine monomers 2.4 R 0 R 1 Yield 2.4a H t Bu 71% 2.4b H Bn 39% 2.4c Me CH 2 (1 Naph) 60% 2.4d Ph i Bu 60% 2.4e Ph Bn 87% 2.4f Ph t Bu 83% 2.4g Ph CH 2 (1 Naph) 40% 2.4h NH 2 Bn 91% 2.4i NH 2 t Bu 75% 2.4j OMe Bn 30% as the catalyst. The double bond of compound 2.3 has Z as its dominate configuration ; probably due to the steric hindrance of the carbonyl group. Th e -unsaturated -

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43 cyanoketones 2.3 react ed with a variety of commercially available formamidine/amidines/guanidine to form the pyrimidine monomers 2.4ag. This methodology has very broad application, in that a library of monomers have been synthesized (Table 2.2). 2.2.3 Conversion of the 5-cyano group to an amidine The key step of the synthesis is the conversion of the 5-cyano to an amidine group. Several methods have been explored, including the Pinner reaction, the thioPinner reaction (Balo et al., 2007), ammonia in methanol (Balo et al., 2007), and NaHMDS or LiHMDS (Bruning, 1997) used as nucleophiles, followed by hydrolysis; but these methods were not successful in yielding the target compounds. Since th e 4-alkyl side chain of our molecules 2.4 (Scheme 2.3) made the cyano group sterically hindered, all the regular methodologies failed. We eventually found a two-step procedure to achieve this proce ss (Scheme 2.4) (Judkins et al., 1996). First, free hydroxylamine was the a nucleophile to attack the cyano group at elevated temperatures to form amidinoximes q ; the N,O single bond was then cleaved in the second step by Scheme 2.4: Synthetic strategy to convert sterically hindered nitriles to amidines

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44 hydrogenation to prepare the amidine r. This hydroxylamine nucleophilic addition reaction is very unique (Scheme 2.4, n to q ), because a great amount of oxygen attacking amide byproduct (yields: 20-50%) was detected (Scheme 2.5). Typically, the neutral nitrogen atom is a lot more nucleophilic than the neutral oxygen atom. Scheme 2.5: Reaction and side reaction of hydroxylamine nucleophilic addition Exact mechanistic details for this reaction have not been reported. However, a possible explanation for the dim in ish ed selectivity between the Nand Onucleophilic species due to the steric hindrance from the ortho substituent on the pyrimidine rings. It should be pointed out that hydroxylamine behaves not only as a nucleophile, but also as a cyano activator. In fact, we found that under the same reaction conditions without hydroxylamine, the cyano group cannot be hydrolyzed to the amide s Further evidence in support of properties in this reaction is that, when using O protected hydroxylamine, (e.g. O -benzylhydroxylamine and O -methylhydroxylamine) not even nitrogen attack product q was formed; while all the starting materials remained. The mechanism proposed by Judkins et al, who originally published the hydroxylamine,

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45 suggested that the hydrogen atom on the oxygen might behave as a Brnsted acid interacting with the nitrogen atom of the cyano group, thus activating it through a fivemembered ring transition state (Scheme 2.6). Scheme 2.6: Proposed transition state of hy droxylamine nucleophilic addition 2.2.4 Synthesis of the pyrimidine dimer Five pyrimid in ylene dimer analogs (Table 2.3) were prepared by condensing the amidines with the -unsaturated -cyanoketones (Scheme 2.7). The same reaction Scheme 2.7: Synthesis of the pyrimidine dimer

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46 conditions which were used to synthesi ze the pyridine monomers were also used to synthesize the dimers. This provides a good example of the broad scope of this condensation methodology to synthesize the pyrimidine rings. Table 2.3: Analogs of the synthesized pyrimidine dimers R 0 R 1 R 2 2.6a Ph Bn CH 2 (1 N aph) 2.6b Ph i Bu CH 2 (1 N aph) 2.6c Ph CH 2 (1 N aph) Bn 2.6d Me CH 2 (1 N aph) Bn 2.6e Me CH 2 (1 N aph) t Bu 2.2.5 X-Ray crystal structure of a representative dimer The X-ray diffraction crystal structure of compound 2.6d (Figure 2.4) was obtained by Dr. Froncezk (Louisiana State University). As it can be seen, the pyrimidine scaffold is arranged in a twisted conformation. This was expected for compounds designed to mimic an -helix. Figure 2.4 Crystal structure of pyrimidine dimer

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47 2.2.6 Synthesis of the pyrimidine trimers Exactly the same process used to synthesize the dimers was also applied to the synthesis of the pyrimidine trimers (Scheme 2.8). Three trimer analogs were synthesized (T able 2.4). Scheme 2.8 Synthesis of the pyrimidine trimers Table 2.4: Analogs of the synthesized pyrimidine trimers R 0 R 1 R 2 R 3 2.8 a Ph i Bu CH 2 (1 N aph) i Bu 2.8 b Ph Bn CH 2 (1 N aph) i Bu 2.8 c Ph CH 2 (1 N aph) Bn i Bu

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48 2.3 Conclusion We have successfully synthesized several 1,4-terpyrimid in ylene analogs. The Xray crystal structure did confirm their potential as -helical mimetics. The newly synthesized series of molecules has been the one that has the closest structural similarity to MDM2 inhibitor. However, none of our molecules are active against p53/MDM2. We reason ed th at although they mimic the side chains of phenylene quite well, our molecules are unable to establish the polar interactions that are available to due to the polar groups present at the head and the tail position. Those hydrogen bonding/salt bridge bonding could be crucial for the molecules bioactivities, as well. Another drawback of our molecules is that they ar e still too hydrophobic to be suitable drug candidates. There is an it is still not enough. Third, the synthesis is a long, linear, time-consuming process; it is very hard to develop a large library of compounds with such a method. In summary, we have successfully developed a new series of non-peptidic helical mim et ics; however, further improvement is needed for this series of molecules to be more efficient p53/MDM antagonists, and be sufficiently drug like, cell permeable compounds. 2.4 Experimental Section 2.4.1 Materials and Methods

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49 Starting materials, organic and inorganic reagents (ACS grade), and solvents were obtained from commerc i al sources and used as received unless otherwise noted. Moistureand air-sensitive reactions were carried out under an atmosphere of argon. Thin layer chromatography (TLC) was performed on glass plates precoated with 0.25 mm thickness of silica gel (60 F-254) with fluorescent indicator (EMD). Column chromatographic purification was performed using silica gel 60 #70-230 mesh (Selecto Scientific). Automated flash chromatography was performed in a FlashMaster II system (Argonaut-Biotage) using Biotage silica cartridges. 1 H NMR and 13 C NMR spectra were obtained using a 400 MHz Varian Mercury or 250 MHz Broker plus instrument at 25 C in chloroform-d (CDCl 3 ), unless otherwise indicated. Chemical shifts (ppm) are reported in parts per million (ppm) relative to internal tetramethylsilane (TMS) or chloroform (ppm 7.26) for 1 H NMR and chloroform (ppm 77.0) for 13 C NMR. Multiplicity is expressed as (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, or m = multiplet) and the values of coupling constants ( J ) are given in Hertz (Hz). High Resolution Mass Spectrometry (HRMS) spectra were carried out on an Agilent 1100 Series in the ESI-TOF mode. Microwave reactions were performed in a closed vessel in a Biotage Initiator I microwave reactor. Melting points (uncorrected) were determined using a Mel-Temp II, Laboratory Devices, MA, USA. 2.4.2 Experimental Procedures Methyl 2-(naphthalen-2-yl)acetate (2.1a)

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50 To a solution of 2-naphthylacetic acid (5.4 mmol) in methanol (10 ml) was added concentrated sulfuric acid (0.3 m mo l). The reaction mixture was stirred under reflux for 3 h. The reaction mixture was concentrated under reduced pressure, and was then extracted between dichloromethane and water. The organic layer was extracted once with saturated sodium bicarbonate solution, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford compound 2.1a as a colorless oil (1.07 g, 100%). 1 H NMR (400 MHz, CDCl 3 ) ppm 3.75 (s, 3H), 3.84 (s, 2H), 7.39 7.56 (m, 3H), 7.77 (s, 1H), 7.80 7.90 (m, 3H). 13 C NMR (100 MHz, CDCl 3 ) ppm 41.6, 52.3, 126.07, 126.4, 127.6, 127.9, 127.9, 128.2, 128.5, 131.7, 132.7, 133.7, 172.3. 3-Oxo-4-phenylbutanenitrile (2.2a) To a solution of methyl phenylacetate (71.0 mmol) and anhydrous acetonitrile (107.2 mmol) in anhydrous THF (20 mL) at 0 C in an ice-bath under an argon atmosphere was added potassium tert -pentoxide (107.2 mmol). The reaction mixture was allowed to warm up to room temperature and stirred under an argon atmosphere for overnight. A precipitate was formed within a few minutes (3-5 min.) of stirring and the reaction mixture remained cloudy until completion. The mixture was filtered and the solid was washed thoroughly with hexanes. The filtered residue was then transferred to a

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51 separatory funnel and was acidified to pH= ~2-3 with saturated aq. potassium bisulfate solution. DCM was then used to extract the desired compound. The organic layer was washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to afford pure 2.2a as a yellow oil (7.34 g, 65%) 1 H NMR (400 MHz, CDCl 3 ) ppm 3.46 (s, 2H), 3.86 (s, 2H), 7.21 7.40 (m, 5H). 13 C NMR (100 MHz, CDCl 3 ) ppm 31.1, 49.2, 113.5, 127.9, 129.3, 129.4, 131.9, 195.1. es-CO 4-(Naphthalen-1-yl)-3-oxobutanenitrile (2.2b) Compound 2.2b was prepared following the procedure described for 2.2a. Isolated yield: 64% yield, yellow solid. 1 H NMR (400 MHz, CDCl 3 ) ppm 3.41 (s, 2H), 4.31 (s, 2H), 7.437.66 (m, 4H), 7.81 7.98 (m, 3H). 13 C NMR (100 MHz, CDCl 3 ) ppm 31.2, 47.8, 113.7, 123.3, 125.9, 126.6, 127.5, 128.7, 129.0, 129.3, 129.4, 132.0, 134.3, 196.0. 4-(Naphthalen-2-yl)-3-oxobutanenitrile (2.2c) Compound 2.2c was prepared following the procedure described for 2.2a. Isolated yield: 71% yield, yellow solid. 1 H NMR (400 MHz, CDCl 3 ) ppm 3.51 (s, 2H), 4.06 (s, 2H), 7.34 (dd, J = 1.8, 8.4 Hz, 1H), 7.51 7.57 (m, 2H), 7.74 (s, 1H), 7.84 7.93 (m, 3H). 13 C

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52 NMR (100 MHz, CDCl 3 ) ppm 31.4, 49.6, 113.8, 126.7, 126.9, 127.1, 127.9, 128.0, 128.8, 129.4, 129.5, 132.9, 133.7, 195.5. 5-Methyl-3-oxohexanenitrile (2.2d) Compound 2.2d was prepared by the same procedure described for 2.2a. Isolated yield: 73%, yellow oil. 1 H NMR (400 MHz, CDCl 3 ) ppm 0.95 (d, J = 0.85 Hz, 3H), 0.97 (d, J = 0.85 Hz, 3H), 2.11-2.25 (m, 1H), 2.49 (d, J = 6.88 Hz, 2H), 3.43 (s, 2H). 13 C NMR (100 MHz, CDCl3) ppm 22.5, 24.7, 32. 6, 51.1, 113.9, 197.2. 3-( 1H -Indol-3-yl)-3-oxopropanenitrile (2.2e) Compound 2.2 was prepared following the procedure described by Radwan, M. A. A. et. al. Isolated yield: 29%, pale orange solid, m.p. 238 o C (lit.= 240 o C). Spectral data is in agreement with literature ( Bioorganic Med. Chem. Lett. 2007, 15, 1206). 2-((Dimethylamino)methylene)-3-oxo-4-phenylbutanenitrile (2.3a)

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53 To a solution of 2.2a (45.8 mmol) in anhydrous THF (24 mL) was added DMF-DMA (59.8 mmol). The reaction mixture was stirred at r.t. for overnight. The reaction mixture was then concentrated under reduced pressure to afford 2.3a as a yellow solid (>90%). The product was used in the next step without further purification. For characterization purpose, the product was recrystallized in ethanol to afford light yellow needle-like crystals, m.p. 134-138 C. 1 H NMR (400 MHz, CDCl 3 ) ppm 3.20 (s, 3H), 3.38 (s, 3H), 3.95 (s, 2H), 7.21 7.34 (m, 5H), 7.80 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 9.0, 46.5, 48.2, 120.5, 126.9, 128.7, 129.8, 129.8, 129.9, 135.0, 158.0, 192.9. HRMS (ESI) calcd. for C 13 H 14 N 2 O [M + H] + 215.1184, found 215.1194. pt-BR 2((D imethylamino)methylene) 4(naphthalen 1yl) 3oxobutanenitrile (2.3b) Compound 2.3b was prepared following the procedure described for 2.3a. Isolated yield: >90%, yellow solid, m.p. 121 122 o C. 1 H NMR (400 MHz, CDCl 3 ) ppm 3.17 (s, 3H), 3.41 (s, 3H), 4.43 (s, 2H), 7.407.54 (m, 4H), 7.77 (d, J = 7.9 Hz, 1H), 7.807.86 (m, 2H), 7.99 (d, J = 8.2 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 39.1, 44.1, 48.1, 80.1, 120.6, 124.6, 125.7, 125. 8, 126.3, 127.9, 128.7, 128.8, 131.8, 132.6, 134.4, 158.1, 192.6. HRMS (ESI) calcd. for C 17 H 16 N 2 O [M + H] + 265.1335, found 265.1313. 2((Dimethylamino)methylene)-4-(naphthalen-2-yl)-3-oxobutanenitrile (2.3c)

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54 Compound 2.3c was prepared following the procedure described for 2.3a. Isolated yield: >90%, yellow solid. 1 H NMR (400 MHz, CDCl 3 ) ppm 3.20 (s, 3H), 3.39 (s, 3H), 4.12 (s, 2H), 7.407.50 (m, 3H), 7.777.84 (m, 5H). 13 C NMR (100 MHz, CDCl 3 ) ppm 39.0, 46.7, 48.2, 80.1, 120.5, 125.8, 126.2, 127.8, 128.0, 128.0, 128.3, 128.5, 132.6, 133.8, 158.0, 192.9. HRMS (ESI) cal cd. for C 17 H 16 N 2 O [M + H] + 265.1335, found 265.1333. 2((Dimethylamino)methylene)-5-methyl-3-oxohexanenitrile (2.3d) Compound 2.3d was prepared following the procedure described for 2.3a. Isolated yield: >90%, yellow solid. 1 H NMR (400 MHz, CDCl 3 ) ppm 0.95 (d, J = 1.48 Hz, 3H), 0.96 (d, J = 1.47 Hz, 3H), 2.13-2.25 (m, 1H), 2.54 (dd, J = 7.03 Hz, 2H), 3.24 (s, 3H), 3.40 (s, 3H), 7.82 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.8, 25.8, 39.0, 48.1, 48.8, 80.8, 120.6, 157.6, 195.4. HRMS (ESI) calcd. for C 10 H 17 N 2 O [M + H] + 181.1341, found 181.1333. pt-BR 2((D imethylamino)methylene) 4,4dimethyl 3oxopentanenitrile (2.3e)

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55 Compound 2.3e was prepared following the procedure described for 2.3a. Isolated yield: >90%, white solid, m.p. 48-49 C. 1 H NMR (400 MHz, CDCl 3 ) ppm 1.32 (s, 9H), 3.23 (s, 2H), 3.42 (s, 2H), 7.92 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 27.0, 39.1, 43.8, 48.5, 121.5, 160.5, 200.4. HRMS (ESI) calcd. for C 10 H 16 N 2 O [M + H] + 181.1341, found 181.1340. 4-( tert -Butyl)pyrimidine-5-carbonitrile (2.4a) To a solution of 2.3e (2.8 mmol) and formamidine acetate salt (16.7 mmol) in anhydrous ethanol (12 mL) was added sodium ethoxide (16.7 mmol). The reaction mixture was stirred under reflux for 24 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10 ) affords 2.4b as a colorless oil (0.32 g, 71%). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.52 (s, 9H), 8.90 (s, 1H), 9.24 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 28.8, 39.9, 107.3, 116.3, 159.4, 162.0, 179.6. 4-Benzylpyrimidine-5-carbonitrile (2.4b)

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56 Compound 2.4b was prepared following the procedure described for 2.4a Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.4b as a colorless oil (39%). 1 H NMR (400 MHz, CDCl 3 ) ppm 4.25 (s, 2H), 7.17 7.26 (m, 2H), 7.267.33 (m, 3H), 8.83 (s, 1H), 9.20 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 43.1, 109.2, 114.8, 127.8, 129.2, 129.5, 135.7, 160.4, 160.5, 172.0. HRMS (ESI) calcd. for C 12 H 9 N 3 [M + H] + 196.0875, found 196.0868. 2-Methyl-4-(naphthalen-1-ylmethyl)pyrimidine-5-carbonitrile (2.4c) Compound 2.4c was prepared following the procedure described for 2.4a, using commercially available acetamidine hydrochloride. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:4) affords 2.4c as an off-white solid (60%). 1 H NMR (400 MHz, CDCl 3 ) ppm 2.75 (s, 3H), 4.73 (s, 2H), 7.58 7.41 (m, 4H), 7.84 (dd, J = 8.1, 23.5 Hz, 2H), 8.27 (d, J = 8.4 Hz, 1H), 8.80 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 26.9, 40.7, 106.2, 115.4, 124.4, 125.7, 126.1, 126.6, 128.6, 128.6, 129.0, 132.1, 132.2, 134.17, 160.6, 171.1, 171.5. 4i so -Butyl-2-phenylpyrimidine-5-carbonitrile (2.4d)

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57 Compound 2.4d was prepared following the procedure described for 2.4a, using commercially available benzamidine hydrochloride. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.4d c as an off-white solid (60%), m.p. 68-69 C. 1 H NMR (400 MHz, CDCl 3 ) ppm 1.05 (d, J = 6.7 Hz, 6H), 2.39 (m, 1H), 2.93 (d, J = 7.2 Hz, 2H), 7.49 7.59 (m, 3H), 8.50 8.53 (m, 2H), 8.93 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.6, 28.9, 45.7, 106.7, 115.8, 129.0, 129.4, 132.4, 136.4, 160.4, 165.7, 173.3. HRMS (ESI) calcd. for C 15 H 15 N 3 [M + H] + 238.1344, found 238.1337. 4-Benzyl-2-phenylpyrimidine-5-carbonitrile (2.4e) Compound 2.4e was prepared following the procedure described for 2.4a, using commercially available benzamidine hydrochloride. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:7) affords 2.4e as an off-white solid (87%), m.p. 138-141 C. 1 H NMR (400 MHz, CDCl 3 ) ppm 4.37 (s, 2H), 7.27 7.30 (m, 1H), 7.35 (dd, J = 10.1, 4.6 Hz, 2H), 7.45 7.59 (m, 5H), 8.49 8.54 (m, 2H), 8.92 (s, 1H).

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58 13 C NMR (100 MHz, CDCl 3 ) ppm 43.3, 105.9, 115.7, 127.6, 129.0, 129.1, 129.5, 129.5, 132.6, 136.1, 136.2, 160.8, 166.0, 171.9. HRMS (ESI) calcd. for C 18 H 13 N 3 [M + H] + 272.1188, found 272.1196. pt-BR 4tert -Butyl-2-phenylpyrimidine-5ca rbonitrile (2.4f) Compound 2.4f was prepared following the procedure described for 2.4a, using commercially available benzamidine hydrochloride. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:7) affords 2.4f as an off-white solid (83%), m.p. 101-103 C. 1 H NMR (400 MHz, CDCl 3 ) ppm 1.60 (s, 9H), 7.49 7.58 (m, 3H), 8.51 8.55 (m, 2H), 8.94 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 28.8, 40.2, 104.0, 117.2, 128.9, 129.4, 132.4, 136.5, 162.5, 164.8, 179.3. HRMS (ESI) calcd. for C 15 H 15 N 3 [M + H] + 238.1344, found 238.1347. 4(N aphthalen-1-ylmethyl)-2-phenylpyrimidine-5-carbonitrile (2.4g)

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59 Compound 2.4g was prepared following the procedure described for 2.4a, using commercially available benzamidine hydrochloride. Purification by flash column ch romatography on silica gel (ethyl acetate/hexane, 1:7) affords 2.4g as an off-white solid (40%). 1 H NMR (400 MHz, CDCl 3 ) ppm 4.83 (s, 2H), 7.44 7.63 (m, 7H), 7.82 (d, J = 8.2, 1H), 7.87 (d, J = 8.1, 1H), 8.37 8.45 (m, 3H), 8.94 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 40.7, 106.2, 115.8, 124.7, 125.8, 126.1, 126.5, 128.6, 128.6, 129.0, 129.0, 129.4, 132.6, 161.0, 165.8, 171.6. 2-Amino-4-benzylpyrimidine-5-carbonitrile (2.4h) Compound 2.4h was prepared following the procedure described for 2.4a, using commercially available guanidine hydrochloride. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:1) affords 2.4h as an off-white solid (91%), m.p. 129-132 C. 1 H NMR (400 MHz, CDCl 3 ) ppm 4.10 (s, 2H), 5.58 (br s, 2H), 7.24-7.38 (m, 5H), 8.45 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 42.9, 97.8, 116.4, 127.5, 129.0, 129.5, 136.0, 162.4, 162.8, 173.5. HRMS (ESI) calcd. for C 12 H 10 N 4 [M + H] + 211.0984, found 211.0972. 2-Amino-4tert -butylpyrimidine-5-carbonitrile (2.4i)

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60 Compound 2.4i was prepared following the procedure described for 2.4a, using commercially available guanidine hydrochloride. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:1) affords 2.4i as an off-white solid (75%), m.p. 89-91 C. 1 H NMR (400 MHz, CDCl 3 ) ppm 1.43 (s, 9H), 5.56 (br s, 2H), 8.44 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 28.5, 39.5, 95.6, 118.3, 162.4, 164.0, 181.2. HRMS (ESI) calcd. for C 9 H 12 N 4 [M + H] + 177.1140, found 177.1148. 4-Benzyl-2-methoxypyrimidine-5-carbonitrile (2.4j) Compound 2.4j was prepared following the procedure described for 2.4a, using commercially available O -methylisourea sulfate. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:3) affords 2.4j as an off-white solid (30%). 1 H NMR (400 MHz, CDCl 3 ) ppm 4.07 (s, 3H), 4.22 (s, 2H), 7.26 (t, J = 7.3 Hz, 1H), 7.29 7.35 (m, 2H), 7.41 (d, J = 7.4 Hz, 2H), 8.68 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 43.1, 56.1, 101.9, 115.5, 127.7, 129.1, 129.5, 135.8, 163.4, 166.2, 175.1. 4'-Benzyl-4-(naphthalen-1-ylmethyl)-2'-phenyl-[2,5'-bipyrimidine]-5-carbonitrile (2.6a)

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61 Step 1: To a solution of compound 2.4e (4.1 mmol) in ethanol (9 mL) was added a solution of hydroxylamine hydrochloride (16.3 mmol ) and potassium carbonate (8.1 mmol) in water (5 mL). The reaction mixture was stirred under reflux overnight. The reaction mixture was concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the intermediate amidoxime, which was used in the next step without further purification. Step 2: To a solution of the intermediate from step 1 in glacial acetic acid (10 mL) was added acetic anhydride (246 L). After 5 min. of stirring, a solution of potassium formate prepared in situ from potassium carbonate (10 mmol) and formic acid (20 mmol) in methanol (4 mL) was added, followed by 10% Pd/C (0.4 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered through Celite TM and rinsed with methanol. The filtrate was concentrated under reduced pressure to afford a yellow residue. This residue was redissolved in DCM and filtered through the Celite TM and rinsed with DCM. The filtrate was concentrated under reduced pressure to

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62 afford the crude the carboxamidine acetate salt as a yellow solid, which was used in the next step without further purification. Step 3: To the solution of the carboxamidine salt from step 2 in ethanol (5 mL) was added compound 2.3d (4.1 mmol) and sodium ethoxide (4.1 mmol). The reaction mixture was stirred under reflux for overnight. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed by reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:5) affords 2.4b as white solid (32%), m.p. 165 166 o C 1 H NMR (400 MHz, CDCl 3 ) ppm 4.45 (s, 2H), 4.84 (s, 2H), 6.88 6.95 (m, 2H), 7.06 7.11 (m, 3H), 7.42 7.56 (m, 7H), 7.84 (dd, J = 8.1, 19.3 Hz, 2H), 8.17 (d, J = 8.2 Hz, 1H), 8.45 8.52 (m, 2H), 9.01 (s, 1H), 9.33 (s, 1H). 13 C NMR ( 100 MHz, CDCl 3 ) ppm 40.1, 43.2, 105.0, 106.3, 115.1, 124.3, 125.5, 125.9, 126.2, 126.5, 127.3, 127.4, 127.7, 128.8, 128.9, 129.0, 129.1, 129.4, 131.6, 132.4, 134.0, 135.1, 135.6, 137.0, 160.3, 160.6, 165.1, 169.1, 172.1. H RMS (ESI) cal c d. f or C 33 H 22 N 5 [M + H] + 490.2026, found 490.2002. 4'iso -Butyl-4-(naphthalen-1-ylmethyl)-2'-phenyl-[2,5'-bipyrimidine]-5-carbonitrile (2.6b)

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63 Compound 2.6b was prepared following the procedure described for 2.6a using 2.4d Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.6c as an off-white solid (33%). 4-Benzyl-4'-(naphthalen-1-ylmethyl)-2'-phenyl-[2,5'-bipyrimidine]-5-carbonitrile (2.6c) Compound 2.6c was prepared following the procedure described for 2.6a using 2.4g Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.6c as an off-white solid (41%). 1 H NMR (400 MHz, CDCl 3 ) ppm 4.15 (s, 2H), 5.17 (s, 2H), 6.96 (d, J = 7.1 Hz, 1H), 7.21 (d, J = 1.9 Hz, 4H), 7.40 7.53 (m, 5H), 7.71 (d, J = 8.2 Hz, 1H), 7.83 7.88 (m, 1H), 8.10 8.15 (m, 1H), 8.39 (ddd, J = 6.8, 3.8, 2.0

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64 Hz, 2H), 8.87 (s, 1H), 9.52 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 40.0, 43.2, 106.3, 115.1, 124.3, 125.5, 125.8, 126.2, 126.6, 127.3, 127.4, 127.7, 128.8, 129.9, 129.4, 160.3, 160.6, 165.1, 160.6, 165.1, 169.1, 172.1. 4-Benzyl-2'-methyl-4'-(naphthalen-1-ylmethyl)-[2,5'-bipyrimidine]-5-carbonitrile (2.6d) Compound 2.6d was prepared following the procedure described for 2.6a using 2.4c Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.6d as an off-white solid (30%) 1 H NMR (400 MHz, CDCl 3 ) ppm 2.81 (s, 3H) 4.04 (s, 2H), 5.08 (s, 2H), 6.76 (d, J = 7.10 Hz, 1H), 7.06-7.11 (m, 2H), 7.14-7.23 (m, 4H), 7.45-7.53 (m, 2H), 7.66 (d, J = 8.21 Hz, 1H), 7.81 (d, J = 8.26 Hz, 1H), 8.06 (d, J = 7.73 Hz, 1H), 8.77 (s, 1H), 9.36 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 26.3, 39.7, 43.1, 106.5, 115.0, 123.9, 125.5, 126.0, 126.1, 126.3, 127.3, 127.5, 127.7, 129.0, 129.1, 129.4, 132.2, 133.9, 134.9, 135.6, 159.7, 160.6, 164.9, 169.0, 169.6, 172.0. 4-( tert -Butyl)-2'-methyl-4'-(naphthalen-1-ylmethyl)-[2,5'-bipyrimidine]-5carbonitrile (2.6e)

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65 Compound 2.6d was prepared following the procedure described for 2.6a using 2.4c Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.6d as an off-white solid (30%). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.35 (s, 9H), 2.77 (s, 3H), 5.11 (s, 2H), 6.80 (d, J = 7.1 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H), 7.43 7.51 (m, 2H), 7.64 (d, J = 8.3 Hz, 1H), 7.78 7.83 (m, 1H), 8.02 (d, J = 9.0 Hz, 1H), 8.80 (s, 1H), 9.32 (s, 1H). 13 C NMR (100 MHz, CDCl3) ppm 14.421.3, 26.4, 28.6, 29.9, 39.7, 40.1, 60.6, 104.6, 116.4, 124.0, 125.4, 125.9, 126.2, 126.2, 127.3, 127.8, 128.9, 132.2, 133.9, 134.7, 159.7, 162.2, 164.1, 168.6, 169.5, 171.3, 179.6. -Cyano-diisobutyl(1 -naphthylmethyl)-2-phenylterpyrimidine (2.8a)

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66 Compound 2.8a was prepared following the procedure described for 2.6a using 2.6b Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.8a as an off-white solid (20%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.71 (d, J = 6.7 Hz, 6H), 0.76 (d, J = 6.6 Hz, 6H), 1.892.01 (m, 1H), 2.05-2.18 (m, 1H), 2.66 (d, J = 7.3 Hz, 2H), 2.95 (d, J = 7.0 Hz, 2H), 5.14 (s, 2H), 6.95 (d, J = 7.0 Hz, 1H), 7.19-7.25 (m, 1H), 7.39-7.47 (m, 5H), 7.65 (d, J = 8.2 Hz, 1H), 7.79 (dd, J = 6.2, 3.3 Hz, 1H), 7.98 (dd, J = 6.1, 3.4 Hz, 1H), 8.41-8.48 (m, 2H), 8.86 (s, 1H), 9.21 (s, 1H), 9.53 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.4, 22.7, 28.4, 29.0, 40.1, 44.5, 45.6, 107.5, 115.0, 124.0, 125.5, 126.0, 126.4, 126.7, 127.5, 127.7, 128.5, 128.8, 128.8, 129.1, 131.1, 132.3, 134.1, 134.7, 137.8, 159.5, 160.0, 160.2, 164.1, 164.5, 164.9, 169.1, 170.0, 173.8. HRMS (ESI) cal cd. for C 38 H 35 N 7 [M + H] + 590.3027, found 590.2984. -Cyano-isobutyl(1 -naphthylmethyl)-4(2 -phenylmethyl)-2phenylterpyrimidine (2.8b):

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67 Compound 2.8a was prepared following the procedure described for 2.6a using 2.6b Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.8b as an off-white solid (20%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.71 (d, J = 6.7 Hz, 6H), 0.76 (d, J = 6.6 Hz, 6H), 1.892.01 (m, 1H), 2.05-2.18 (m, 1H), 2.66 (d, J = 7.3 Hz, 2H), 2.95 (d, J = 7.0 Hz, 2H), 5.14 (s, 2H), 6.95 (d, J = 7.0 Hz, 1H), 7.19-7.25 (m, 1H), 7.39-7.47 (m, 5H), 7.65 (d, J = 8.2 Hz, 1H), 7.79 (dd, J = 6.2, 3.3 Hz, 1H), 7.98 (dd, J = 6.1, 3.4 Hz, 1H), 8.41-8.48 (m, 2H), 8.86 (s, 1H), 9.21 (s, 1H), 9.53 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.4, 22.7, 28.4, 29.0, 40.1, 44.5, 45.6, 107.5, 115.0, 124.0, 125.5, 126.0, 126.4, 126.7, 127.5, 127.7, 128.5, 128.8, 128.8, 129.1, 131.1, 132.3, 134.1, 134.7, 137.8, 159.5, 160.0, 160.2, 164.1, 164.5, 164.9, 169.1, 170.0, 173.8. HRMS (ESI) cal cd. for C 38 H 35 N 7 [M + H] + 590.3027, found 590.2984. -Cyano-isobutyl(2 -phenylmethyl)-4(1 -naphthylmethyl)-2phenylterpyrimidine (2.8C):

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68 Compound 2.8c was prepared following the procedure described for 2.6a using 2.6c Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:10) affords 2.8c as an off-white solid (17%) 1 H NMR (400 MHz, CDCl 3 ) ppm 0.93 (d, J = 6.6 Hz, 6H), 2.13 2.27 (m, 1H), 2.85 (d, J = 7.2 Hz, 2H), 4.55 (s, 2H), 5.10 (s, 2H), 6.94 7.08 (m, 5H), 7.21 (t, J = 7.6 Hz, 1H), 7.29 7.40 (m, 6H), 7.62 (d, J = 8.2 Hz, 1H), 7.75 (dd, J = 6.2, 3.3 Hz, 1H), 8.06 (dd, J = 5.9, 3.4 Hz, 1H), 8.23 8.28 (m, 2H), 8.92 (d, J = 0.5 Hz, 1H), 9.38 (d, J = 0.5 Hz, 1H), 9.44 (d, J = 0.5 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.6, 29.2, 39.7, 42.2, 45.8, 107.6, 115.0, 124.8, 125.6, 125.6, 126.0, 126.7, 127.0, 127.2, 127.5, 128.2, 128.6, 128.7, 128.7, 128.9, 129.3, 131.2, 132.7, 134.0, 135.5, 137.4, 138.1, 159.9, 160.1, 160.2, 164.2, 164.5, 164.5, 168.6, 169.1, 173.7. 2.5 References Balo, C., Lopez, C., Brea, J. M., Fernandez, F., and Caamano, O. (2007). Synthesis and evaluation of adenosine antagonist activity of a series of [1,2,4]triazolo[1,5c]quinazolines. Chemical & Pharmaceutical Bulletin 55, 372-375. Bruning, J. (1997). Lithium and potassium bis(trimethylsilyl)amide: utilizing nonnucleophile bases as nitrogen sources. Tetrahedron Letters 38, 3187-3188. Davis, J. M., Truong, A., and Hamilton, A. D. (2005). Synthesis of a 2,3';6',3''Terpyridine Scaffold as an alpha -Helix Mimetic. Organic Letters 7, 5405-5408. Ernst, J. T., Becerril, J., Park, H. S., Yin, H., and Hamilton, A. D. (2003). Design and application of an alpha -helix-mimetic scaffold based on an oligoamide-foldamer strategy: Antagonism of the Bak BH3/Bcl-xL complex. Angewandte Chemie, International Edition 42, 535-539. Estroff, L. A., Incarvito, C. D., and Hamilton, A. D. (2004). Design of a Synthetic Foldamer that Modifies the Growth of Calcite Crystals. Journal of the American Chemical Society 126, 2-3. Ji, Y., Trenkle, W. C., and Vowles, J. V. (2006). A high-yielding preparation of beta keto nitriles. Organic Letters 8, 1161-1163.

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69 Judkins, B. D., Allen, D. G., Cook, T. A., Evans, B., and Sardharwala, T. E. (1996). A versatile synthesis of amidines from nitriles via amidoximes. Synthetic Communications 26, 4351-4367. Moisan, L., Odermatt, S., Gombosuren, N., Carella, A., and Rebek, J., Jr. (2008). Synthesis of an oxazole-pyrrole-piperazine scaffold as an alpha -helix mimetic. European Journal of Organic Chemistry, 1673-1676. Reuman, M., Beish, S., Davis, J., Batchelor, M. J., Hutchings, M. C., Moffat, D. F. C., Connolly, P. J., and Russell, R. K. (2008). Scalable Synthesis of the VEGF-R2 Kinase Inhibitor JNJ-17029259 Using Ultrasound-Mediated Addition of MeLi-CeCl3 to a Nitrile. Journal of Organic Chemistry 73 1121-1123. Rodriguez, J. M., and Hamilton, A. D. (2006). Intramolecular hydrogen bonding allows simple enaminones to structurally mimic the i, i +4, and i +7 residues of an alpha -helix. Tetrahedron Letters 47, 7443-7446. Volonterio, A., Moisan, L., and Rebek, J., Jr. (2007). Synthesis of pyridazine-based scaffolds as alpha -helix mimetics. Organic Letters 9, 3733-3736. Yin, H., Lee, G.-i., Park, H. S., Payne, G. A., Rodriguez, J. M., Sebti, S. M., and Hamilton, A. D. (2005a). Terphenyl-based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition 44, 2704-2707. Yin, H., Lee, G.-i., Sedey, K. A., Kutzki, O., Park, H. S., Orner, B. P., Ernst, J. T., Wang, H. -G., Sebti, S. M., and Hamilton, A. D. (2005b). Terphenyl-Based Bak BH3 alpha Helical Proteomimetics as Low-Molecular-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society 127, 10191-10196.

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70 Chapter Three: Convergent Approach to Synthesis 2,5-Terpyrimidinylene Based Derivatives 3.1 Introduction 3.1.1 Optimization of synthetic route to the 2,5-terpyrimidinylene scaffold We have successfully synthesized several analogs of the 2,5-terpyrimidinylene scaffold; however, the design and the synthesis need fu rther optimizations. 3.1.2 Modifications that might lead to improvement of the bioactivities Figure 3.1: -terphenylene derivatives

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71 Figure 3.2: Synthesized 2,5-terpyrimidinylene derivatives Our strategy focused on the synthesis of novel analogs of 1,4terphenylene derivatives (Figure 3.1); however, the 2,5-terpyrimidinylene analogs (Figure 3.2) that we have synthesized did not show any activities again st p53/MDM2. Figure 3.3: Structure comparison of 1,4-terphenylene and 2,5-pyrimidinylene scaffold

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72 We proposed the following two reasons. First, comparison of our synthesized (Figure 3.3) show that, even though the side chains of our molecules mimic the side chains of his 1,4-terphenylene derivatives our molecules lack of the hydrogen bonding / salt bridge potential that feature at the extreme terminals in From of the 1,4-terphenylene derivatives (Orner et al., 2001) which were used to target the interaction between calmodulin (CaM) and an Rhelical domain of smooth muscle myosin light-chain kinase (smMLCK) later use of the se derivatives to target the interaction between p53 and MDM2 (Yin et al., 2005) the importance of the terminal carboxylic acid groups. In all cases, molecules without the terminal carboxylic acid groups did not show bio-activities. Our 2,5-terpyrimidinylene scaffold has a phenyl group, and a cyano group at the terminal positions, which can not be involved in hydrogen bonding / salt bridge interactions at physiology pH. This lack of interaction might be one of the major reasons why our molecules did not show bioactivity. We attempted to mimic the terminal hodology that we have developed, it was very challenging. We were also -cyano group to carboxylic acid, but because of the high steric hindrance of the 5-position, and the high hydrophobicity of the 2,5-terpyrimidinylene derivative, the hydrolysis was very challenging. Furthermore, we suggest that more hydrophilic elements be added to our molecules. The solubility of our molecules in DMSO was not as good as we planned.

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73 3.1.3 Problems existing in the developed synthetic process It is difficult to make a library of molecules using the most recent synthesis process that has been developed because it is a long and tedious process. This procedure is a linear synthesis, containing 9 steps, almost all of which required at least 12 h reaction time Typically, it took over a week to make one analog. Scheme 3.1: Reaction and side reaction of hydroxylamine nucleophilic addition Additionally, the overall yield is very low, normally about 1-2%. The low yield of the overall synthesis was the result of two reactions. The first is the hydroxylamine nucleophilic addition reaction, which generally produces 20-50% side product amide b (Scheme 3.1). The second yield limiting reaction is the condensation reaction to form the pyrimidine ring (Scheme 3.2). This reaction generates water in situ which hydrolyzed Scheme 3.2: Reaction and side reaction of amidine condensation reaction

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74 the limiting reagent amidine d to amide f ; this side product is confirmed by proton NMR. This may be the reason why we never had a yield greater than 50% when we did the condensation reaction to make the pyrimidine dimers and trimers. Unfortunately, both of th e se reactions need to be carried out twice during the overall synthesis, which made the overall yield of the synthesis low. To simplify the synthesis process, we choose to make our 2,5-terpyrimidine molecules with a phenyl group at the 2-position. We could very well use more diverse groups, e.g. NH 2 etc., but that would increase the difficulty of synthesis even more. 3.1.4 Convergent synthetic strategy To improve the yield of the synthesis, and to reduce the synthesis time, we chose to use a convergent strategy (Scheme 3.3) instead of the linear synthesis. Retrosynthetic analysis revealed that the target molecule g could be derived from the amidine h and the Scheme 3.3: Retrosynthesis using the convergent strategy

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75 -unsaturated -pyrimidineketone i The synthesis of amidine h has been previous ly reported; so we focused our attention on exploring the synthesis of -unsaturated pyrimidineketone i A convergent synthetic approach presents several advantages. First, the amidine h and the -unsaturated -pyrimidineketones i can be simultaneously synthesized, reducing the overall synthesis time. Second, the synthesis of several analogs h and i would allow us to easily achieve the diversity of the library of the target molecules. Last but not least, since the top amidine half h would be easily made, we can use more than 1 equiv. of the amidine h in the last condensation step to increase overall yield. 3.2 Results and Discussion for the synthesis of the new unsaturated ketones 3.2.1 Retrosynthetic to make the -unsaturated -pyrimidineketones The synthesis of the fragment i (Scheme 3.4) relied on our well-developed reaction: -substituted ketone j reacting with DMF-DMA. The -pyrimidine ketone j in turn, could be made by condensation of -amidine ketone k with another -unsaturated Scheme 3.4: Retrosynthesis of the -unsaturated -pyrimidineketones

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76 -cyanoketone (Scheme 3.2 c) And the -amidine ketone k could be prepared using our extensive collection of -cyano ketone l 3.2.2 Attempted synthesis of the -unsaturated -pyrimidineketones 3.5 With this in mind, the following steps have been carried out (Scheme 3.5). Containing a good electronphilic ketone group, starting material 3.1 was protected to prevent the side reactions. We decided to protect the ketone group by reducing it to alcohol, because the alcohol group should be inert to the following reaction conditions which contain both acidic and basic conditions. Reduction of the starting material cyano ketone 3.1 using sodium borohydride at 0 C gave the -cyano alcohol 3.2 in excellent yield. To convert cyano group 3.2 to amidine 3.3, we relied on the Scheme 3.5: Attempted synthesis of compound 3.5

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77 hydroxylamine reaction. Nucleophilic addition of hydroxylamine to compound 3.2 at high temperature gave the amidoxime 3.3 in very good yield. It is worth to point out that, in this reaction (Scheme 3.6), no amide side product was observed. This shows the influence of the steric hindrance in this reaction: if the cyano group is not hindered, only the nitrogen atom of the hydroxylamine behaves as the nucleophile. After this reaction, hydrogenation of the amidoxine 3.3 gave the amidine intermediate, which condensed with an -unsaturated -cyanoketone 2.3d to give the -pyrimidine alcohol 3.4. However, at this stage, we could not oxidize the 2 o alcohol 3.4 to the ketone 3.5. We tried Jones oxidation (Zibuck and Streiber, 1993), Swern oxidation (Dondoni and Perrone, 2000), Dess-Martin oxidation (Sniady et al., 2007), and PCC (Tu et al., 2003). However, only the Dess-Martin oxidation gave a trace mount of product, no product could be detected in the other oxidation methods. Based on the results, no starting material alcohol could be recycled after all these oxidation methods. We reason ed that the pyrimidine ring is not stable under those oxidation conditions. Scheme 3.6: Hydroxylamine addition of compound 3.2 3.2.3 Attempted synthesis of -unsaturated ketones using 1,2,3-triazole ring

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78 Some new results from our group showed that, besides the six-membered ring, the five-membered ring could also be used in the scaffold while retaining the -helical mimetic properties. Therefore, we modified our target molecules by replacing the pyrimidine ring with the 1,2,3-triazole ring (the detailed reason will be discussed in Chapter 4). The synthetic strategy remained unvaried (Scheme 3.7); the 1,2,3-triazole ring could be prepared using the azide alkyne Huisgen cycloaddition, also known as click chemistry. Scheme 3.7: Synthesis of the -unsaturated -triazoleketones

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79 First, a simple bromination reaction at the -position of the ketone gave the bromine ketone 3.6CH 2 N 2 strategy, but it is a simple one step high yielding reaction, perfect for the trial series. Then, azide behaved as a nucleophile to undergo a S N 2 reaction to kick out the bromide as a leaving group. The resulting azide 3.7 underwent an azide alkyne Huisgen cycloaddition to form the -triazole ketone 3.8 (Chen et al., 2007). This -triazole ketone 3.8 reacted with DMF-DMA at room temperature resulting in compound 3.9, a n analog of the -unsaturated ketone i in Scheme 3.3. To check the reactivity of this series of -unsaturated -triazoleketones, 3.9 was used to condense with guanidine. A s expected, a pyrimidine ring was formed, getting an 80% yield in the cyclization reaction. Scheme 3.8: Condensation to synthesize the hybrid trimer 3.11

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80 After the proof of the reactivity of the -unsaturated -triazoleketones we condensed 3.9 with an amidine monomer 2.5d to make a hybrid pyrimidine-triazole trimer molecule 3.11 (Scheme 3.8). 3.2.4 Biological testing of 3.10 Compound 3.10 was tested in FP assay against p53/MDMx interaction, in which it showed 6 17% activities at 100 L. Considering 3.10 is a dimer molecule which has a molecular weight of only 324.38, this scaffold showed the potential to be an antagonist of p53/MDMx interaction. 3.3 Conclusion We have successfully developed a convergent route to synthesize a new generation of non-peptidic -helix mim et ics. The newer generation of the molecules has more hydrogen bonding/ionic bonding potentials at the top and bottom positions, while retaining the hydrophobic side chains that mimic the side chains of the -helical mimet ics. The synthesis of this new generation of trimers has been simplified from the 2,5-terpyrimidinylene generation; it contains 7 steps from the longest route, and most of those reactions are high yielding. This strategy is more conductive in synthesizing a library of molecules. 3.4 Experimental Procedures 3-Hydroxy-4-phenylbutanenitrile (3.2)

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81 To a solution of 2.2a (0.47 mmol) in methanol (2 ml) cooled to 0 C in an ice-bath was added sodium borohydride (0.73 mmol). The reaction mixture was stirred at 0 C for 1.5 h. After the reaction, aq. H 2 SO 4 (1 M) was added to the reaction mixture until pH = 6. The mixture was then concentrated under reduced pressure. The residue was extracted with ethyl acetate 3 times. The combined organic layer was extracted once with brine solution, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:2) to afford 3.2 as a white solid (70 mg, 92%). 1 H NMR (400 MHz, CDCl 3 ) ppm 2.50 (qd, J = 16.7, 5.5 Hz, 2H), 2.69 (bs, 1H), 2.88 (d, J = 6.7 Hz, 2H), 4.07 4.18 (m, 1H), 7.19 7.37 (m, 5H). 13 C NMR (100 MHz, CDCl 3 ) ppm 25.3, 43.0, 68.8, 117.9, 127.4, 129.1, 129.6, 136.6. N',3-Dihydroxy-4-phenylbutanimidamide acetate (3.3) To a solution of compound 3.2 (3.9 mmol) in ethanol (11 mL) was added a solution of hydroxylamine hydrochloride (19.7 mmol) and potassium carbonate (9.8 mmol) in water (4 mL). The reaction mixture was stirred under reflux for 14 h. The reaction mixture was concentrated under reduced pressure. The residue was extracted between DCM and

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82 water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as yellow solid. Purification by flash column chromatography on silica gel (methanol/ethyl acetate, 1:20) affords the amidoxime 3.3 as a white solid (0.45 g, 59%). 1 H NMR (400 MHz, CDCl 3 ) ppm 2.14 2.40 (m, 2H), 2.69 2.78 (m, 2H), 4.00 4.10 (m, 1H), 4.80 5.30 (br m, 4H), 7.13 7.32 (m, 5H). 13 C NMR (100 MHz, CDCl 3 ) ppm 37.8, 43.7, 70.7, 126.8, 128.8, 128.8, 129.7, 138.2. HRMS (ESI) cal cd. for C 10 H 14 N 2 O 2 [M + H] + 195.1128, found 195.1203. 2(2 -Hydroxy-3-phenylpropyl)-4-isobutylpyrimidine-5-carbonitrile (3.4) Step 1: To a solution of the 3.3 (1.5 mmol) in glacial acetic acid (3 mL) was added acetic anhydride (175 L). After 5 min. of stirring, a solution of potassium formate prepared in situ from potassium carbonate (5 mmol) and formic acid (10 mmol) in methanol (2 mL) was added followed by 10% Pd/C (0.2 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through Celite TM and rinsed with methanol. The filtrate was concentrated under reduced pressure to afford a yellow residue. This residue was redissolved in DCM and filtered through Celite TM and rinsed with DCM. The filtrate was concentrated under reduced pressure to afford the crude

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83 amidine acetate salt as a yellow solid, which was used in the next step without further purification. Step 2: To the solution of the amidine salt (0.8 mmol) from step 2 in ethanol (3 mL) was added compound 2.3d (0.8 mmol) and triethylamine (1.6 mmol). The reaction mixture was stirred under reflux for overnight. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed by reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:2) afforded 3.4 as white solid (32%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.98 (d, J = 6.7 Hz, 6H), 2.23 (tt, J = 13.7, 6.9 Hz, 1H), 2.84 (d, J = 7.3 Hz, 2H), 2.90 (dd, J = 15.9, 5.9 Hz, 1H), 2.98 (d, J = 7.0 Hz, 1H), 3.12 (dd, J = 16.3, 9.0 Hz, 1H), 3.23 (dd, J = 16.3, 2.9 Hz, 1H), 3.97 ( br s, 1H), 4.43 (d, J = 8.3 Hz, 1H), 7.207.36 (m, 5H), 8.80 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.5, 22.5, 29.2, 43.5, 45.2, 45.6, 70.9, 107.2, 115.1, 126.8, 128.8, 129.8, 138.1, 160.0, 171.8, 173.2 2-Bromo-1-phenylethanone (3.6) To an ice cooled solution of acetophenone (1.7 mmol) and catalytic amount of aluminum chloride in diethyl ether (4 ml) was added bromine (4.3 mmol). The reaction mixture was stirred at 0 C until the red color faded. After the reaction, aq. H 2 SO 4 (1 M) was added to the reaction mixture until pH = 6. The mixture was then concentrated under

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84 reduced pressure. The residue was extracted with ethyl acetate 3 times. The combined organic layer was extracted once with brine solution, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:2) affords 3.6 as a white solid (70 mg, 92%). 1 H NMR (400 MHz, CDCl 3 ) ppm 2.50 (qd, J = 16.7, 5.5 Hz, 2H), 2.69 (bs, 1H), 2.88 (d, J = 6.7 Hz, 2H), 4.07 4.18 (m, 1H), 7.197.37 (m, 5H). 2-Azido-1-phenylethanone (3.7) Sodium azide (4.3 mmol) was dissolved in DMSO (9 mL) and the solution was stirred for 12 h. To this solution was added 3.6 (2.2 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was then concentrated under reduced pressure using the Biotage V10. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:7) affords the 3.7 as a colorless oil (0.15 g, 59%). 1 H NMR (400 MHz, CDCl 3 ) ppm 4.56 (s, 2H), 7.46 7.54 (m, 2H), 7.59 7.66 (m, 1H), 7.88 7.92 (m, 2H). 2(4 (1 -Hydroxy-3-methylbutyl)-1H-1,2,3-triazol-1-yl)-1-phenylethanone (3.8)

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85 To a solution of 3.7 (0.29 mmol) and 5-methyl-1-hexyn-3-ol (0.29 mmol) in tert -butanol (4 mL) was added a solution of (+)-sodium L-ascorbate (0.29 mmol) in water (2 mL) and copper(II) sulfate pentahydrate (0.14 mmol) in water (2 mL), respectively. The reaction mixture was stirred at room temperature for 24 h. The mixture was then concentrated under reduced pressure. The residue was extracted between ethyl acetate and water. The combined organic layer was extracted once with brine solution, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 2:1) affords 3.8 as a white solid (63 mg, 79%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.93 (d, J = 6.3 Hz, 6H), 1.67 (ddd, J = 4.2, 9.1, 13.3 Hz, 1H), 1.81 (qd, J = 6.1, 13.2 Hz, 2H), 3.36 (br s, 1H), 4.95 (dd, J = 5.0, 8.5 Hz, 1H), 5.81 (s, 2H), 7.49 (dd, J = 4.8, 10.7 Hz, 2H), 7.60 (s, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.94 (dd, J = 1.1, 8.3 Hz, 2H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.2, 23.4, 24.7, 46.4, 55.7, 65.3, 122.8, 128.4, 129.4, 134.1, 134.8, 152.5, 190.8. HRMS (ESI) calcd. for C 15 H 19 N 3 O 2 [M + H] + 274.1550 found 274.1557. 3-(Dimethylamino)-2(4 (1 -hydroxy-3-methylbutyl)-1H-1,2,3-triazol-1-yl)-1phenylprop-2en -1-one (3.9)

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86 To a solution of 3.8 (1.6 mmol) in THF (24 ml) under argon atmosphere was added DMF-DMA (4.3 mmol). The reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure affords the crude product as brown solid. Purification by flash column chromatography on silica gel (methanol/ethyl acetate, 1:20) afforded 3.9 as a white solid (0.42 g, 82%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.90 (dd, J = 6.2, 2.8 Hz, 6H), 1.55 1.80 (m, 3H), 2.32 (br s, 3H), 3.13 (br s, 3H), 4.90 (dd, J = 8.1, 5.2 Hz, 1H), 7.29 (s, 1H), 7.30 (s, 1H), 7.33 7.43 (m, 4H), 7.56 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.3, 23.3, 24.7, 46.6, 65.4, 108.9, 126.6, 127.9, 128.4, 130.7, 139.2, 150.1, 152.1, 189.5. HRMS (ESI) cal cd. for C 18 H 24 N 4 O 2 [M + H] + 329.1972, found 329.1972. 1(1 (2 -Amino-4-phenylpyrimidin-5-yl)-1H-1,2,3-triazol-4-yl)-3-methylbutan-1-ol (3.10)

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87 To a solution of 3.9 (0.2 mmol) in ethanol (3 mL) under argon atmosphere was added guanidine hydrochloride (0.5 mmol) and sodium ethoxide (0.5 mmol). The reaction mixture was stirred under reflux for 15 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed by reduced pressure affords the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 4:1) affords 3.10 as white solid (80 %). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.91 (dd, J = 6.3, 1.7 Hz, 6H), 1.471.78 (m, 3H), 7.117.49 (m, 5H), 7.76 (s, 1H), 8.37 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 21.5, 21.99, 24.4, 46.3, 64.5, 104.99, 121.1, 124.8, 128.0, 135.2, 152.6, 156.6, 162.8, 164.0. HRMS (ESI) cal cd. for C 17 H 20 N 6 O [M + H] + 325.1771, found 325.1763. 1(1 -(4'iso Butyl-2',4-diphenyl-[2,5'-bipyrimidin]-5-yl)-1H-1,2,3-triazol-4-yl)-3methylbutan-1-ol (3.11)

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88 To a solution of 2.5 (0.4 mmol), freshly made using the method described before, in ethanol (8 mL) under argon atmosphere was added 3.9 (0.2 mmol) and triethylamine (0.4 mmol). The reaction mixture was stirred under reflux for 14 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed by reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:1) affords 3.10 as white solid. 1 H NMR (400 MHz, CDCl 3 ) ppm 0.94 0.98 (m, 12H), 1.64 1.80 (m, 4H), 2.10 2.16 (m, 1H), 2.31 2.43 (m, 1H), 4.95 5.03 (m, 1H), 7.38 7.41 (m, 5H), 7.51 7.55 (m, 4H), 8.55 8.59 (m, 2H), 9.09 (s, 1H), 9.48 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.3, 22.9, 23.2, 24.8, 28.5, 45.0, 46.6, 65.5, 122.8, 128.1, 128.8, 131.3, 137.6, 153.1, 155.4, 159.6, 164.4, 169.9. HRMS (ESI) cal cd. for C 31 H 33 N 7 O [M + H] + 520.2819, found 520.2810. 3.5 References Chen, H., Taylor, J. L., and Abrams, S. R. (2007). Design and synthesis of beta methoxyacrylate analogues via click chemistry and biological evaluations. Bioorganic & Medicinal Chemistry Letters 17, 1979-1983. Dondoni, A., and Perrone, D. (2000). Synthesis of 1,1-dimethylethyl (S)-4-formyl-2,2dimethyl-3-oxazolidinecarboxylate by oxidation of the alcohol. Organic Syntheses 77, 64-77. Orner, B. P., Ernst, J. T., and Hamilton, A. D. (2001). Toward Proteomimetics: Terphenyl Derivatives as Structural and Functional Mimics of Extended Regions of an alpha -Helix. Journal of the American Chemical Society 123, 5382-5383.

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89 Sniady, A., Morreale, M. S., and Dembinski, R. (2007). Electrophilic cyclization with Niodosuccinimide: preparation of 5(4 -Bromophenyl)-3-iodo-2(4 -methylphenyl)furan. Organic Syntheses 84, 199-208. Tu, Y., Frohn, M., Wang, Z.-X., and Shi, Y. (2003). Synthesis of 1,2:4,5-di-oisopropylideneD -erythro-2,3-hexodiulo-2,6-pyranose. A highly enantioselective ketone catalyst for epoxidation. Organic Syntheses 80, No pp given. Yin, H., Lee, G.-i., Park, H. S., Payne, G. A., Rodriguez, J. M., Sebti, S. M., and Hamilton, A. D. (2005). Terphenyl-based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition 44, 2704-2707. Zibuck, R., and Streiber, J. (1993). Ethyl 3-oxo-4-pentenoate (Nazarov's reagent). Organic Syntheses 71, 236-242.

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90 Chapter Four: Design and Synthesis of Hybrid Scaffold Non-peptidic -Helical Mimetics 4.1 Introduction 4.1.1 General scaffold of non-peptidic -helical mimetics We have previously synthesized several analogs of 2,5-terpyrimidinylene scaffold molecules, which aimed at mimic king the most in vitro active non-peptidic p53-MDM2 inhibitor (Yin et al., 2005a) that has been reported so far. Nevertheless, the reported nonpeptidic -helical mim et ics have more diverted structures (Figure 2.2). A common feature of those molecules is the presence of three directly bonded rings as the backbone, positioned in a linear configuration. The ring fragments could be either regular covalent-bonded rings (Davis et al., 2005), (Volonterio et al., 2007) (Moisan et al., 2008) or rings formed by hydrogen bond (Ernst et al., 2003), (Estroff et al., 2004), (Yin et al., 2005b), (Rodriguez and Hamilton, 2006). They are preferably aromatic rings, for the benefit of the flat geometrical conformation associated. Each ring bears an alkyl or aromatic side chain designed to mimic the side chains of the original peptides. It is crucially that all alkyl or aromatic side chains point to the same face, mimicking the i i + 4(3) i + 7 positions of the peptides on the same face of the helix. An

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91 abstract model that stands for this series of non-peptide -helical mim et ics is shown in Figure 4.1. Figure 4.1: Abstract structure of -helical mim et ics 4.1.2 New library design strategy From previous reports and studies, it was clear that it is very challenging to find good p53/MDM2 antagonists: a favorable scaffold, favorable side chains, and a favorable order of side chains all affect the activities of the antagonists. Thus, our strategy relied on easily buildable scaffolds, amenable to library development, allowing the introduction diverse side chains as well as terminal polar groups. 4.1.3 Introduction of the hybrid scaffold The majority of non-peptidic -helical mimetics used iterative design, e.g. a, b c in Figure 2.1 (Yin et al., 2005a), (Ernst et al., 2003), (Davis et al., 2005). The benefits are the following: the alkyl or aromatic side chains are ensured to point to the same

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92 direction, and the synthesis is easy to design. However, iterative design usually involves iterative linear synthesis, which is multistep and can be low yielding. As long as we can make sure the side chains of the scaffold are pointed to the same direction, we could consider the scaffold a good choice. We decided to focus on hybrid scaffolds, featuring five-membered and six-membered aromatic rings for the simplification of synthesis. A pyrimidine core was our six-membered rings candidate of choice, for its promising properties shown in the previous projects carried out in our lab. Of the many aromatic five-membered rings, we chose to use 1,2,3-triazole rings. The 1,2,3-triazole rings have several characteristics suiting our needs. First, it could be easily synthesized from azides and acetylenes (Chen et al., 2007), by the high yielding azide-alkyne Huisgen cycloaddition, and the azides and acetylenes are readily with diverse sructures to ensure the diversity of the 1,2,3-triazole rings. Second, the introduction of five-membered ring with three nitrogen atoms would lower the logP value of the molecules (see discussion s of Table 2. 1) I n other words, the synthesized molecules have a higher water-solubility potential if the scaffold contains more nitrogen atoms; thus, 1,2,3-triazole is a better scaffold candidate than pyrimidine rings for the benefit of water solubility. 4.1.4 Strategy to extend the pyrimidine rings using Huisgen cycloaddition 1,2,3-triazole ring could be made from the reaction between azides and acetylenes. If we can attach either an azide group or an acetylene group to the pyrimidine ring, we could easily make a dimer (Scheme 4.1). With this in mind, the following dimer synthesis has been carried out as a test of the 1,2,3-triazole ring strategy.

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93 Scheme 4.1: Synthetic strategy to make the hybrid dimer 4.2 Results and Discussion for introducing 1,2,3-triazole to the scaffold 4.2.1 Synthesis of 4-triazole pyrimidine hybrid dimer 4.6 The same procedure that was used to generate pyrimidine monomer 2.4 was applied to the synthesis of monomer 4.2 (Scheme 4.2), bearing a hydrogen in place of a cyano group at the 4-position. Further, we reacted the 4-alkyl group of compound 4.5 with an alkyl azide 4.7 to attach a 1,2,3-triazole ring to the 4-position of the pyrimidine ring.

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94 Scheme 4.2: Synthesis of hybrid dimer 4.6

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95 In the first step, we react ed 1,1,1,-trifluoro-5-methyl-2,4-hexanedione with DMFDMA to form an -unsaturated ketones 4.1. This is an interesting reaction, because the product of the reaction is dependent on the electronegativity properties of the group adjacent R 1 (Scheme 4.3). If it is an alkyl group, R 1 will stay in the final molecule; on the other hand, if R 1 is a strong electron-withdrawing group, e.g. CF 3 then the whole R 1 CO group will be eliminated from the final molecules. Scheme 4.3: Comparison of different substituted 1,3-diketones reacting with DMF-DMA The mechanism of this reaction has not been studied, a possible mechanism is proposed in Scheme 4.4.

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96 Scheme 4.4: One possible mechanism of 1,3-diketone reacting with DMF-DMA The tautomer of 1,1,1,-trifluoro-5-methyl-2,4-hexanedione a acts like a nucleophile to attack the iminium salt formed after DMF-DMA eliminates one methoxide. After the formation of the tetrahedral intermediate c a second methoxide will be kicked out by the lone pair electron on the nitrogen atom to form the intermediate d There are two possible reactions for the intermediate d If R 1 is a regular alkyl group,

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97 a regular E2 reaction will take place, the -proton will be eliminated to form the product e However, if the R 1 group is a strong enough electron-withdrawing group, the adjacent carbonyl group would be activated so it can be a good electrophile towards MeO/MeOH. After the attack of MeO/MeOH, the carbon-carbon bond on the other side of the carbonyl group will be broken, that pair of electrons will become the pi electrons of a double bond. One way to prove this mechanism is that, in this case, methyl trifluoroacetate should be a side product. After the -unsaturated ketone 4.1 was prepared, it underwent a cyclization with guanidine to form a pyrimidine ring (Scheme 4.2). This reaction occurs under the same condition we utilized for the -unsaturated -cyanoketones even though the -unsaturated ketone 4.1 is less reactive due to the lack of the electron withdrawing cyano group. Then an iodination at the least electron deficient position of the pyrimidine ring (Shepherd and Fellows, 1948), the 5-position, gave us the iodide compound 4.3 The iodide compound 4.3 underwent Sonogashira cross-coupling reaction (Chen et al., 2007), coupling an acetone-protected acetylene to the pyrimidine ring. Deprotection of the acetylene using potassium hydroxide in toluene at elevated temperature gave us the unprotected acetylene product 4.5 (Watanabe et al., 2009). This is the synthesis of the acetylene half for the Huisgen cycloaddition. For the azide half, we should be able to use any alkyl azides or azide derivatives of amino acids. We used benzyl azide 4.7 for it is easy synthesis (Alvarez and Alvarez, 1997).

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98 Finally, Huisgen cycloaddition between 4.5 and 4.7 under standard condition gave the 1,2,3-triazole 4.6 in good yield. 4.2.2 Benefits of the 1,2,3-triazole ring The polarity of dimer 4.6 is the highest among all the non-peptidic -helical mimetics dimers that we have synthesized. The synthesis to attach a 1,2,3-triazole ring to the 4-position of a pyrimidine ring is easy and high yielding. The combination of the two factors makes the 1,2,3-triazole ring modified scaffold promisin g. 4.2.3 Synthesis of 2-triazole pyrimidine hybrid dimer 4.13 From the discussion above, we have successfully developed a way to attach a 1,2,3-triazole ring to the 4-position of the pyrimidine rings. We also explored the possibility of attaching a 1,2,3-triazole ring to the 2-position of the pyrimidine rings. To form a 1,2,3-triazole ring at the 2position of the pyrimidine ring, we can either attach an azide group or an acetylene group. Initial attempts to replace the 2chloride on the pyrimidines with actylide anion failed. The acetylene amidine underwent self-polymerization, therefore, cannot be used as the starting material to synthesize the pyrimidines. Therefore, a better option is to attach an azide group to the 2-position of the pyrimidine rings. Again, direct replacement of 2-chloro pyrimidine with azide derivatives did not produce any product, which was inconvenient also because of the difficulty of synthesizing the 2-chloro-4-alkyl-pyrimidines (Scheme 4.8). In the end, we

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99 deploy ed the reaction of hydrazine reacting with nitrous acid to synthesis the azide; and successfully attached a 1,2,3-triazole ring to the 2-position of pyrimidine rings. We found that DMF-DMA can react with all kinds of ketones besides the -cyano ketones, as long as the -position contains at least two hydrogen atoms (Scheme 4.5). If the -position is attached to some electron-withdrawing group, e.g. cyano group, nitro group, 1,2,3,-triazole group, this reaction occurs at room temperature; whereas higher temperatures are required for less activated ketones. Scheme 4.5: General methodology to synthesize -unsaturated ketones from ketones The -unsaturated ketone 4.9d reacted with protected aminoguanidine in the microwave giving the pyrimidine ring product 4.10 (Scheme 4.6). The hydrazine group of 4.10 is protected as imine (Martins et al., 2004), which is normally acid labile. However, this imine group is very stable, as a result of the conjugated system, so even concentrated hydrochloride acid and sulfuric acid could not break down the imine group. Luckily, with a large excess of hydrazine hydrate, hydrazine product 4.11 could be formed (Gonzalez, 1988). The reaction to remove the benzylaldehyde protecting group

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100 should be through a competition mechanism, where hydrazine competes with the hydrazine group of 4.10 for the benzylaldehyde. Compound 4.11 reacts with nitrous acid to form a 2-azido pyrimidine 4.12 (Lindsay and Allen, 1942). This azide compound 4.12 reacted with commercially available 5-methyl-1-hexyn-3-ol through Huisgen cycloaddition to form the compound 4.13 which has a 1,2,3,-triazole ring attached at the Scheme 4.6: Synthesis of hybrid dimer 4.13

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101 2-position of the pyrimidine ring. Again, compound 4.13 is as polar as compound 4.6, both are more polar than the pyrimidine dimers that we have synthesized. 4.2.4 Evaluation 1,2,3-triazole ring as a fragment in our scaffold So far, we have proven the possibility of attaching 1,2,3-triazole ring to both 2position (top) and 5-position (bottom) of a pyrimidine ring. Both compounds 4.6 and 4.13 are significantly more polar than the other pyrimidine dimers that we have previously synthesized. Unfortunately, we failed all the attempts to attach iodine at the 5position of 4.13 (Shepherd and Fellows, 1948) (Beierlein et al., 2008), (Guillard and Viaud-Massuard, 2008), thus no trimer of this series has been synthesized yet. The failure of this reaction might be the result of the switch from the electron-donating group NH 2 to electron-withdrawing 1,2,3-triazole ring. Nevertheless, the 1,2,3-triazole ring incorporation showed promising result toward our goal of water-soluble non-peptidic -helical mimetic library design and synthesis. 4.3 Results and Discussion for introducing amino acids to the scaffold 4.3.1 Possibility of introducing amino acids to the scaffold Even though most of the libraries that have been developed so far feature three flat rings (mostly aromatic) that have been attached linearly (Figure 2.1) (Yin et al., 2005a), (Davis et al., 2005), (Moisan et al., 2008), (Volonterio et al., 2007), this might

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102 not be an essentia l requirement to this series of non-peptidic -helical mimetics. Co mputational studies showed that the side chain of the 2-amino acid on the pyrimidine ring is at the right position, i + 7 (Figure 4.2). Figure 4.2: Trimer with an amino acid in the scaffold fitting the abstract scaffold There are several advantages of introducing an amino acid to the scaffold. Its side chain is in the right position; introduction of amino acid will not increase the overall lipophilicity of the molecule (it should decrease the lipophilicity for amino acids do not contain the aromatic system in the backbone ), and the nitrogen carbon bond that connects the amino acid to the pyrimidine ring will not be labile like the peptide bond. Most imp ortantly, it is very convenient to introduce an amino acid to the pyrimidine ring by aromatic substitution reactions; thus the introduction of amino acids to the scaffold fits our need to simplify the synthesis. 4.3.2 Testing of the new scaffold

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103 Again, we would like to keep the second fragment of our scaffold a pyrimidine ring. This is because it is relatively easy to introduce new groups to the 2and 5positions of 4-alkyl pyrimidines. Between the 2and 5-position, we decided to put the amino acids in the 2-position. For the third fragment of our scaffold, we decided to use a 1,2,3-triazole, because of its promising properties that we have proven before. The pyrimidine ring will have an electron-donating nitrogen atom at the 2-position, so it would be possible to connect a 1,2,3-triazole ring to the 5-position. Our new scaffold is shown in Figure 4.3. Figure 4.3 : Possible conformation of the new scaffold 4.3.3 Retrosynthesis of the trimer

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104 The target molecule g could be synthesized from the dimer h using the method developed (Scheme 4.7). Compound h could, in turn, be synthesized from the 2-chloro4-alkyl-pyrimidine i Scheme 4.7: Retrosynthesis of the hybrid trimer Unfortunately, compound i is relatively challenging to synthesize. For the two methods that we have tried (Scheme 4.8), neither one gave us promising results. We first tried the diazonium salt route to convert a NH 2 group to a chloride compound 4.14 (Dhanda et al., 1999). This method gave us about a 30% yield, with a lot of starting material remaining. In the other route, we attempted to react aminocyanogen with HCl gas to produce chloroamidine hydrochloride 4.15 in about a 30% yield (Henderson et al., 2006). Then we condensed the chloroamidine 4.15 with the -unsaturated cyanoketone 2.3d to form the 2-chloropyrimidine 4.16. The second step has a 40% yield, so the overall yield to prepare 4.16 is around 12%. In summary, neither one of methods that have been tried provided us promising results to pursue.

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105 Scheme 4.8: Synthesis of the 2-chloro-pyrimidine ring 4.3.4 Modification of the new target scaffold and the retrosynthesis route Some of the molecules that have been developed so far used ether groups instead of alkyl group as the side chains to mimic the side chains of -helices (Figure 2.1) (Ernst et al., 2003) (Estroff et al., 2004), (Yin et al., 2005b). If we modify our target molecule from g to g (Scheme 4.9), the overall geometry of the molecule should not be changed, but the synthesis is greatly simplified. Target molecule g could be synthesized from the dimer h using the strategy that has been well developed (Scheme 4.2). The extra oxygen atom at the 4-position of the pyrimidine ring can further help the iodination reaction of the h molecule. The dimer h could be synthesized by nucleophilic aromatic substitution reaction one step from the monomer i Because of the reactivity difference between the two chlorine atoms on the

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106 commercially available molecule 2,4-dichloropyrimidine, the 4-chloride could be substituted selectively at room temperature to make the monomer i Scheme 4.9: Modification of the target molecule 4.3.5 Synthesis of the trimer 4.21 The synthesis is shown in Scheme 4.10. Benzyl alcohol was deprotonated using sodium hydride, it replaced the 4-chloride of the 2,4-dichloropyrimidine at room temperature (I rie et al., 2008). Trace amounts of 2,4-dibenzyloxylpyrimidine product could be detected, but the mono-substitution reaction product 4-benzyloxylpyrimidine

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107 Scheme 4.10: Synthesis of trimer 4.22

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108 4.17 was the predominate product, with a yield of 78%. Then, a free amino group of an amino acid replaced the 2-chloride at 155 o C under microwave irradiation (Humphries et al., 2009). The yield of this step is relatively low; fortunately, there is no side reaction occurring, allowing us to recover most of the starting material left over. Next, an iodination reaction gave us compound 4.19 in good yield. Subsequently, Sonogashira reaction coupled an ethynyltrimethylsilane to the dimer. We did not use 2-methyl-3butyn-2-ol here, because although we could couple it to our dimer, the potassium hydroxide that was used to deprotect the acetone group in the next step also react ed with another part of the molecule. Therefore, we chose TMS protected acetylene, and then took the TMS group off by TBAF under mild conditions to synthesize the acetylene half 4.20. For the azide reactant, we could very well use the benzyl azide 4.7 previously synthesized. But we decided to test the compatibility of azide derivatives of amino acids with our scafford. The azide derivative of valine 4.22 was synthesized by a reported procedure (Goddard-Borger and Stick, 2007), using 1H-imidazole-1-sulfonyl azide as the azide source, potassium carbonate as base, and copper sulfate pentahydrate as catalyst. The Huisgen cycloaddition between acetylene 4.20 and azide 4.22 went very well, getting almost quantit at ive yield. 4.3.6 Properties of the timer 4.21 Trimer 4.21 has greatly improved water solubility when compared to compound 2.8, and 3.10. Also, trimer 4.17 is synthetically the easiest to prepare among the three

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109 trimer scaffolds ( 2.8, 3.10, and 4.17) that we have developed; its synthesis involves seven steps, six of which are excellent yielding reactions. Due to the good water solubility as well as its amenability to library-synthesis the scaffold of trimer series 4.17 appears to be the most promising -helix mimetics. The bio-activity of compound 4.17 against p53/MDM and Bcl-x L are currently under investigation. 4.4 Biological testing of 4.6 and 4.13 Dimer compounds 4.6 and 4.13 was tested in Enzyme-linked immunosorbent assay (ELISA ) assay against p53/MDM2 interaction (Figure 4.4, 4.6 in blue, 4.13 in yellow). Figure 4.4: Elisa results of 4.6 and 4.13

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110 Dimer 4.13 showed promising results at lower concentration. But its activity remained unchanged after certain concentration. This indicates that there might still be a solubility problem. Dimer 4.6 also showed some activity against p53/MDM2. Considering the sizes of 4.6 and 4.13, we can conclude that our non-peptidic helical scaffolds are properly designed. The activities of trimer 4.21 are under investigation. 4.5 Conclusion The work shown in this chapter provide a new generation of non-peptidic helical mimetics. There are three basic principles that are leading our design. The side chains of our designed molecules should act as mimetics of the side chains of an -helix. Second, our molecules should possess improved water solubility. Third, the molecules should be easy to synthesize to generate a focus ed library. Based on those three criteria we introduced the 1,2,3-triazole rings and amino acids into the scaffold, and those two scaffold segments showed very promising properties towards the principle. Our target molecule has been redesigned, and one analog 4.21 has synthesized. The synthesized molecule is now under biological testing to check bio-activities against p53/MDM and Bak/Bcl-x L 4.6 Experimental Section 1-(Dimethylamino)-4-methylpent-1en -3-one (4.1)

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111 To a solution of commercially available 1,1,1-trifluoro-5-methyl-2,4-hexanedione (7.7 mmol) in THF (5 ml) under argon atmosphere was added DMF-DMA (10.0 mmol). The reaction mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure to afford the crude product as brown solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexanes, 4:1) affords 4.1 as a colorless oil (0.84 g, 77%). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.07 (dd, J = 6.9, 1.7 Hz, 6H), 2.46 2.59 (m, 1H), 2.81 (br s, 3H), 3.00 (br s, 3H), 5.02 (d, J = 12.6 Hz, 1H), 7.54 (d, J = 12.6 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 19.8, 37.2, 39.8, 44.9, 93.9, 152.7, 202.5. 4iso -Propylpyrimidin-2-amine (4.2) To a solution of 4.1 (5.1 mmol) and guanidine hydrochloride (25.3 mmol) in anhydrous ethanol (10 mL) under argon atmosphere was added triethylamine (25.3 mmol). The reaction mixture was stirred in reflux for 24 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash

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112 column chromatography on silica gel (ethyl acetate/hexane, 2:1) affords 4.2 as a white solid (0.41 g, 58%). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.22 (d, J = 7.0 Hz, 6H), 2.77 (hept, J = 6.9 Hz, 1H), 5.09 (br. s, 2H), 6.49 (d, J = 5.2 Hz, 1H), 8.17 (d, J = 5.2 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 21.9, 36.1, 108.6, 158.3, 163.1, 177.4. 5-Iodo-4-isopropylpyrimidin-2-amine (4.3) A suspension of 4.2 (0.4 mmol) and mercury(II) acetate (0.2 mmol) in water (4 mL), was heated to be boiling for 2 min, and a hot solution of iodine (0.4 mmol) in 1,4-dioxane (4 mL) was added. The reaction mixture was stirred at 100 C for 30 min. After the reaction, a mixture of potassium iodide and sodium sulfite was added to the reaction mixture, and the mixture was stirred overnight. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:3) affords 4.3 as a yellow solid (68 mg, 65%). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.17 (d, J = 6.8 Hz, 6H), 3.19 (hept, J = 6.7 Hz, 1H), 5.31 (br. s, 2H), 8.37 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 21.0, 37.5, 81.1, 162.7, 164.9, 176.1. HRMS (ESI) cal cd. for C 7 H 10 IN 3 [M + H] + 263.9992, found 264.0174.

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113 4(2 -Amino-4-isopropylpyrimidin-5-yl)-2-methylbut-3-yn-2-ol (4.4) To a solution of 4.3 (0.3 mmol) and 2-methyl-3-butyn-2-ol (0.3 mmol) in anhydrous acetonitrile (5 mL) under argon atmosphere was added tetrakis(triphenylphosphine)palladium(0) (0.009 mmol), copper(I) iodide (0.02 mmol) and triethylamine (1.5 mL). The reaction mixture was stirred under reflux for 20 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 2:1) affords 4.4 as a white solid (40 mg, 73%). HRMS (ESI) cal cd. for C 12 H 17 N 3 O [M + H] + 220.1444, found 220.1497. 5-Ethynyl-4-isopropylpyrimidin-2-amine (4.5)

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114 To a solution of 4.4 (0.1 mmol) in toluene (5 mL) was added was added potassium hydroxide (0.5 mmol). The reaction mixture was stirred at 70 C for 3 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between dichloromethane and saturated ammonium chloride aqueous solution. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:2) affords 4.5 as a colorless oil (11 mg, 75%). 5(1 -Benzyl-1H-1,2,3-triazol-4-yl)-4-isopropylpyrimidin-2-amine (4.6) To a solution of 4.4 (0.1 mmol) and benzyl azide 4.7 (0.1 mmol) in tert -butanol (2 mL) was added a solution of (+)-sodium L-ascorbate (0.1 mmol) in water (1 mL), and then a solution of copper(II) sulfate pentahydrate (0.01 mmol) in water (1 mL). The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 2:1)

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115 affords 4.6 as a white solid (14 mg, 70 %). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.19 (d, J = 6.7 Hz, 6H), 3.24 3.36 (m, 1H), 5.07 (br. s, 2H), 5.59 (s, 2H), 7.30 7.44 (m, 5H), 7.47 (s, 1H), 8.33 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 21.6, 29.9, 31.8, 54.5, 121.6, 128.3, 129.4, 134.7, 144.0, 158.4, 162.8, 174.7. Benzyl azide (4.7) Sodium azide (10.0 mmol) was dissolved in DMSO (20 mL) and the solution was stirred at room temperature for 12 h. To this solution was added benzyl bromide (5.0 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture wa s then concentrated under reduced pressure using the Biotage V10. The residue was extracted between diethyl ether and water. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a colorless oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:20) affords 3.6 as a colorless oil (0.47 g, 71%). 2-Benzylidenehydrazinecarboximidamide hydrochloride (4.8) To a solution of aminoguanidine hydrochloride (1.1 mmol) in ethanol (2 mL) was added benzaldehyde (1.0 mmol). The reaction mixture was placed in a microwave reactor for

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116 10 min. at 160 C. The reaction mixture was then concentrated under reduced pressure to afford the intermediate 4.8 which was used in the next step without further purification. 4-(Dimethylamino)but-3en -2-one (4.9a) The mixture of acetone (6.8 mmol) and DMF-DMA (6.8mmol) was stirred under reflux for 16 h. The reaction mixture was then concentrated under reduced pressure to afford 4.9a as a yellow solid, which was used in the next step without further purification. 1-(Dimethylamino)-4-methylhex-1en -3-one (4.9b) The mixture of commercially available 3-methyl-2-pentanone (8.1 mmol) and DMFDMA (10.4 mmol) was stirred at 85 C for 16 h. The reaction mixture was then concentrated under reduced pressure to afford 4.9b as a yellow solid, which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 ) ppm 0.81 (t, J = 7.4 Hz, 3H), 1.01 (d, J = 6.9 Hz, 3H), 1.32 (td, J = 14.0, 7.0 Hz, 1H), 1.60 (dq, J = 22.2, 7.4 Hz, 1H), 2.29 (dq, J = 13.8, 6.9 Hz, 1H), 2.87 (s, 6H), 4.98 (d, J = 12.6 Hz, 1H), 7.50 (d, J = 12.6 Hz, 1H). 1-Cyclopropyl-3-(dimethylamino)prop-2en -1-one (4.9c)

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117 The mixture of commercially available cyclopropyl methyl ketone (10.1 mmol) and DMF-DMA (13.4 mmol) was stirred at 85 C for 16 h. The reaction mixture was then concentrated under reduced pressure to afford 4.9c as a yellow solid, which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 ) ppm 0.700.77 (m, 2H), 0.96 1.03 (m, 2H), 1.70 (br. s, 3H), 1.74 1.84 (m, 1H), 5.19 (d, J = 12.6 Hz, 1H), 7.55 (d, J = 12.6 Hz, 1H). 3-(Dimethylamino)-1-phenylprop-2en -1-one (4.9d) The mixture of commercially available acetophenone (4.3 mmol) and DMF-DMA (5.6 mmol) was stirred at 85 C for 16 h. The reaction mixture was then concentrated under reduced pressure to afford 4.9d as a yellow solid, which was used in the next step without further purification. 1 H NMR (400 MHz, CDCl 3 ) ppm 2.92 (br. s, 3H), 3.14 (br. s, 3H), 5.71 (d, J = 12.4 Hz, 1H), 7.34 7.49 (m, 3H), 7.80 (d, J = 12.4 Hz, 1H), 7.86 7.92 (m, 2H). 2(2 -Benzylidenehydrazinyl)-4-phenylpyrimidine (4.10)

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118 To a solution of 4.8 (4.5 mmol) in anhydrous ethanol (3 mL) was added 4.9d (4.4 mmol). The reaction mixture was placed in a microwave reactor for 2 h at 155 C. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between dichloromethane and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:3) affords 4.10 as a yellow solid (0.76 g, 64%). 1 H NMR (400 MHz, CDCl 3 ) ppm 7.20 (d, J = 5.2 Hz, 1H), 7.30 7.39 (m, 3H), 7.45 7.53 (m, 3H), 7.70 (d, J = 6.7 Hz, 2H), 7.79 (s, 1H), 8.08 (dd, J = 6.5, 2.9 Hz, 2H), 8.58 (d, J = 5.1 Hz, 1H), 9.27 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 109.6, 127.4, 127.4, 128.8, 129.1, 131.2, 134.4, 137.0, 142.7, 159.4, 160.2, 165.7. 2-Hydrazinyl-4-phenylpyrimidine (4.11)

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119 To a solution of 4.10 (0.1 mmol) in ethanol (5 mL) was added hydrazine hydrate (5 mL). The reaction mixture was stirred under reflux for 12 h. The reaction mixture was then concentrated under reduced pressure to afford 4.11 as a yellow oil, which was used in the next step without further purification. 2-Azido-4-phenylpyrimidine (4.12) To a solution of 4.11 (0.1 mmol) in acetic acid (0.5 mL) was added a solution of sodium nitrite (0.7 mmol) in water (0.5 mL). The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between dichloromethane and sodium bicarbonate saturated aqueous solution. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:5) affords 4.12 as a white solid (16 mg, 57%). 1 H NMR (400 MHz, CDCl 3 ) ppm 7.47 (d, J = 5.3 Hz, 1H), 7.49 7.57 (m, 3H), 8.11 (dd, J = 7.4, 1.9 Hz, 2H), 8.63 (d, J = 5.2 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 112.7, 127.5, 129.3, 131.9, 135.8, 159.8, 166.4. HRMS (ESI) calcd. for C 10 H 7 N 5 [M + H] + 198.0774, found 198.0808. 3-Methyl-1(1 (4 -phenylpyrimidin-2-yl)-1H-1,2,3-triazol-4-yl)butan-1-ol (4.13)

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120 To a solution of 4.12 (0.1 mmol) and commercially available 5-methyl-1-hexyn-3-ol (0.1 mmol) in tert -butanol (2 mL) was added a solution of (+)-sodium L-ascorbate (0.1 mmol) in water (1 mL), and then a solution of copper(II) sulfate pentahydrate (0.03 mmol) in water (1 mL). The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 3:1) affords 4.13 as a white solid (14 mg, 70%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.95 (d, J = 6.3 Hz, 6H), 1.72 1.86 (m, 3H), 5.06 (dd, J = 8.3, 4.7 Hz, 1H), 7.48 7.54 (m, 3H), 7.71 (d, J = 5.3 Hz, 1H), 8.14 (dd, J = 7.8, 1.8 Hz, 2H), 8.58 (s, 1H), 8.83 (d, J = 5.3 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.2, 23.5, 24.8, 29.9, 45.5, 65.6, 116.2, 119.7, 127.7, 129.5, 132.37, 135.3, 160.0, 166.8. HRMS (ESI) calcd. for C 17 H 19 N 5 O [M + H] + 310.1662, found 310.1688. 4-( tert -butyl)-2-chloropyrimidine-5-carbonitrile (4.14)

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121 To a solution of 2.4i (0.6 mmol) and antimony(III) chloride (1.2 mmol) in anhydrous chloroform (5 mL) under argon atmosphere was added iso -pentyl nitrite (2.0 mmol) very slowly. The reaction mixture was stirred in reflux for 7 h. The reaction mixture was then poured in to a saturated sodium bicarbonate aqueous solution. The mixture was filtered through Celite TM The filtrate was extracted with diethyl ether. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:1 0) affords 4.14 as a colorless oil (30%). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.46 (s, 9H), 8.71 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 28.7, 29.9, 40.3, 105.8, 115.6, 163.0, 164.1, 183.0. Carbamimidic chloride hydrochloride(4.15) To a solution of cyanamide (11.7 mmol) in diethyl ether (5 0 mL) was added HCl (g) for 30 min. The reaction mixture was stirred at room temperature for an additional 5 h. The reaction mixture was filtered through Celite TM The residue was rinsed with lots of diethyl ether to afford the crude product 4.15 as a white so lid (0.40g, 30%), which was

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122 used in the next step without further purification. 1 H NMR (400 MHz, DMSO ) ppm 6.25 (br. s, 2H), 12.17 (br. s, 2H). 13 C NMR (100 MHz, DMSO ) ppm 155.2. 2-Chloro-4-isobutylpyrimidine-5-carbonitrile (4.16) To a solution of 4.15 (0.5 mmol) and 2.3d (0 .4 mmol) in anhydrous 1,4-dioxane (3 mL) under argon atmosphere was added triethylamine (0.5 mmol). The reaction mixture was stirred at reflux for 13 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:3) affords 4.16 as a colorless oil (40%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.98 (d, J = 6.7 Hz, 6H), 2.12 2.25 (m, 1H), 2.66 (d, J = 7.3 Hz, 2H), 5.49 (s, 2H), 8.45 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.6, 22.7, 28.8, 45.6, 116.7, 162.1, 162.9, 175.0. 4(B enzyloxy)-2-chloropyrimidine (4.17)

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123 To a suspension of sodium hydride (12.6 mmol) in anhydrous N,N -dimethylformamide (5 mL) at 0 C in an ice-bath under argon atmosphere was added benzyl alcohol (5.8 mmol) slowly. To this solution was added a solution of 2,4-dichloropyrimidine (5.8 mmol) in tetrahydrofuran. The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:5) affords 4.17 as a white solid (1.0 g, 78%). 1 H NMR (400 MHz, CDCl 3 ) ppm 5.43 (s, 2H), 6.71 (d, J = 5.7 Hz, 1H), 7.36 7.47 (m, 5H), 8.31 (d, J = 5.7 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 69.4, 107.6, 128.8, 128.9, 128.9, 135.4, 159.1, 160.4, 170.3. Methyl 2((4 -(benzyloxy)pyrimidin-2-yl)amino)-4-methylpentanoate (4.18) To a solution of 4.17 (0.8 mmol) and L-leucine methyl ester hydrochloride (1.6 mmol) in anhydrous ethanol (3 mL) was added sodium methoxide (1.6 mmol). The reaction mixture was placed in a microwave reactor for 2 h at 160 C. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between dichloromethane and water. The organic layer was dried over sodium sulfate, and the

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124 solvent was removed under reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:3) affords 4.18 as a white solid (49 mg, 39%). 1 H NMR (400 MHz, CDCl 3 ) ppm0.96 (dd, J = 10.3, 6.5 Hz, 6H), 1.58 1.87 (m, 3H), 3.70 (s, 3H), 4.55 4.69 (d, J = 5.5 Hz, 1H), 5.31 (s, 2H), 6.10 (d, J = 5.6 Hz, 1H), 7.28 7.44 (m, 4H), 8.03 (d, J = 5.2 Hz, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.2, 23.1, 25.1, 41.8, 52.3, 53.3, 67.7, 128.3, 128.7, 136.8, 169.8, 170.4, 174.8. HRMS (ESI) calcd. for C 18 H 23 N 3 O 3 [M + H] + 330.1812 found 330.1852. Methyl 2((4 -(benzyloxy)-5-iodopyrimidin-2-yl)amino)-4-methylpentanoate (4.19) To a suspension of 4.18 (0.2 mmol) and mercury(II) acetate (0.1 mmol) in water (2 mL), that was heated to boiling for 2 min, was added a hot solution of iodine (0.2 mmol) in 1,4-dioxane (2 mL). The reaction mixture was stirred at 100 C for 2 h. After the reaction, a mixture of potassium iodide and sodium sulfite was added to the reaction mixture, and the mixture was stirred overnight. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash

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125 column chromatography on silica gel (ethyl acetate/hexane, 1:5) affords 4.19 as a yellow solid (42 mg, 62%). 1 H NMR (400 MHz, CDCl 3 ) 0.95 (dd, J = 13.5, 6.3 Hz, 6H), 1.53 1.85 (m, 3H), 3.70 (s, 3H), 4.43 4.68 (m, 1H), 5.37 (s, 2H), 7.37 (ddd, J = 25.0, 18.5, 7.5 Hz, 2H), 7.28 7.48 (m, 5H), 8.26 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 22.1, 23.1, 25.1, 52.4, 61.3, 68.6, 127.6, 128.2, 128.7, 136.5, 161.4, 164.8, 167.0, 173.9. Methyl 2((4 -(benzyloxy)-5-ethynylpyrimidin-2-yl)amino)-4-methylpentanoate (4.20) Step 1: To a solution of 4.19 (0.1 mmol) and ethynyltrimethylsilane (0.2 mmol) in anhydrous acetonitrile (3 mL) under argon atmosphere was added te trakis(triphenylphosphine)palladium(0) (0.003 mmol), copper(I) iodide (0.007 mmol) and triethylamine (1 mL). The reaction mixture was stirred under reflux for 20 h. The reaction mixture was then concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (ethyl acetate/hexane, 1:5) to afford the intermediate as a white solid, which was used in the next step without further purification. Step 2: To a solution of the intermediate from step 1 in anhydrous tetrahydrofuran (1 mL) at 0 C in an ice-bath under argon atmosphere was added tetrabutylammonium fluoride

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126 (0.03 mmol). The reaction mixture was stirred at 0 C for 10 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:5) affords 4.20 as a white solid (30 mg, 92%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.791.02 (m, 6H), 1.58 1.83 (m, 3H), 3.27 (s, 1H), 3.69 (s, 3H), 4.46 4.60 (m, 1H), 5.41 (s, 2H), 6.04 (br. s, 1H), 7.31 (d, J = 7.2 Hz, 1H), 7.36 (t, J = 7.3 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 8.21 (s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 23.1, 25.5, 29.9, 41.5, 52.4, 53.3, 61.4, 68.1, 82.4, 127.8, 128.1, 128.7, 136.6, 160.6, 162.3, 169.5, 178.2. HRMS (ESI) cal cd. for C 20 H 23 N 3 O 3 [M + H] + 3541812, found 354.1800. 2(4 (4 -(Benzyloxy)-2-((1-methoxy-4-methyl-1-oxopentan-2-yl)amino)pyrimidin-5yl)-1H-1,2,3-triazol-1-yl)-3-methylbutanoic acid (4.21) To a solution of 4.20 (0.1 mmol) and 4.22 (0.1 mmol) in tert -butanol (1 mL) was added a solution of (+)-sodium L-ascorbate (0.1 mmol) in water (0.5 mL), and then a solution of

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127 copper(II) sulfate pentahydrate (0.05 mmol) in water (0.5 mL). The reaction mixture was stirred at room temperature for 23 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (methanol / dichloromethane, 1:20) affords 4.21 as a white solid (41 mg, 99%). 1 H NMR (400 MHz, CDCl 3 ) ppm 0.86 (dd, J = 32.4, 6.4 Hz, 6H), 1.02 (dd, J = 20.0, 6.8 Hz, 6H), 1.59 1.86 (m, 3H), 2.24 (dq, J = 13.2, 6.7 Hz, 1H), 3.68 (s, 3H), 4.11 4.20 (m, 1H), 4.40 4.54 (m, 1H), 5.37 5.59 (m, 1H), 7.27 7.43 (m, 5H), 8.12 (s, 1H), 8.77 (s, 1H), 10.76 (br. s, 1H). 13 C NMR (100 MHz, CDCl 3 ) ppm 14.3, 14.5, 18.0, 19.77, 25.0, 29.3, 29.7, 31.2, 40.4, 52.5, 53.9, 61.4, 61.5, 68.7, 69.8, 70.0, 103.4, 103.4, 122.8, 128.1, 132.3, 138.5, 149.1, 159.6, 166.4, 172.9, 175.1, 179.9. HRMS (ESI) cal cd. for C 25 H 32 N 6 O 5 [M + H] + 497.2507, found 497.2484. 2-Azido-3-methylbutanoic acid (4.22) To a solution of L-valine (0.06 mmol), potassium carbonate (0.16 mmol), and copper(II) sulfate pentahydrate (0.006 mmol) in methanol (2 mL) was added imidazole-1-sulfonyl azide hydrochloride (0.07 mmol). The reaction mixture was stirred at room temperature for 5 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between ethyl acetate and HCl (1M, aqueous). The organic layer was dried

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128 over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a colorless oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:1) affords 4.21 as a colorless solid (90%). 1 H NMR (400 MHz, CDCl 3 ) ppm 1.01 (d, J = 6.7 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 2.17 2.33 (m, 1H), 3.78 (d, J = 5.7 Hz, 1H), 11.80 (s, 1H). 4.7 References Alvarez, S. G., and Alvarez, M. T. (1997). A practical procedure for the synthesis of alkyl azides at ambient temperature in dimethyl sulfoxide in high purity and yield. Synthesis, 413-414. Beierlein, J. M., Frey, K. M., Bolstad, D. B., Pelphrey, P. M., Joska, T. M., Smith, A. E., Priestley, N. D., Wright, D. L., and Anderson, A. C. (2008). Synthetic and Crystallographic Studies of a New Inhibitor Series Targeting Bacillus anthracis Dihydrofolate Reductase. Journal of Medicinal Chemistry 51, 7532-7540. Chen, H., Taylor, J. L., and Abrams, S. R. (2007). Design and synthesis of beta methoxyacrylate analogues via click chemistry and biological evaluations. Bioorganic & Medicinal Chemistry Letters 17, 1979-1983. Davis, J. M., Truong, A., and Hamilton, A. D. (2005). Synthesis of a 2,3';6',3''Terpyridine Scaffold as an alpha -Helix Mimetic. Organic Letters 7, 5405-5408. Dhanda, A., Knutsen, L. J. S., Nielsen, M.-B., Roberts, S. M., and Varley, D. R. (1999). Facile conversion of 4-endo-hydroxy-2-oxabicyclo[3.3.0]oct-7en -3-one into carbocyclic 2'-deoxyribonucleoside analogs. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 3469-3475. Ernst, J. T., Becerril, J., Park, H. S., Yin, H., and Hamilton, A. D. (2003). Design and application of an alpha -helix-mimetic scaffold based on an oligoamidefo ldamer strategy: Antagonism of the Bak BH3/Bcl-xL complex. Angewandte Chemie, International Edition 42, 535-539. Estroff, L. A., Incarvito, C. D., and Hamilton, A. D. (2004). Design of a Synthetic Foldamer that Modifies the Growth of Calcite Crystals. Journal of the American Chemical Society 126, 2-3.

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129 Goddard-Borger, E. D., and Stick, R. V. (2007). An Efficient, Inexpensive, and ShelfStable Diazotransfer Reagent: Imidazole-1-sulfonyl Azide Hydrochloride. Organic Letters 9, 3797-3800. Gonzalez, A. (1988). Simple preparation of N-alkyl-N-arylhydrazines from diazotable Narylamines. Synthetic Communications 18, 1225-1229. Guillard, J., and Viaud-Massuard, M.-C. (2008). Synthesis and biological evaluations of new pyrrolo[2,3-b]pyrimidine as SDI analogs. Heterocycles 75 1163-1189. Henderson, E. A., Bavetsias, V., Theti, D. S., Wilson, S. C., Clauss, R., and Jackman, A. L. (2006). Targeting the alpha -folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase. Bioorganic & Medicinal Chemistry 14, 5020-5042. Humphries, P. S., Do, Q.-Q. T., and Wilhite, D. M. (2009). Chemically enabled synthesis of 2-amino-4-heteroarylpyrimidines. Tetrahedron Letters 50, 2552-2554. Irie, O., Yokokawa, F., Ehara, T., Iwasaki, A., Iwaki, Y., Hitomi, Y., Konishi, K., Kishida, M., Toyao, A., Masuya, K. et al. (2008). 4-Amino-2-cyanopyrimidines: Novel scaffold for nonpeptidic cathepsin S inhibitors. Bioorganic & Medicinal Chemistry Letters 18, 4642-4646. Lindsay, R. O., and Allen, C. F. H. (1942). Phenyl Azide. Organic Syntheses 22, No pp given. Martins, T., Franca, T., Ramalho, T. C., and Figueroa-Villar, J. D. (2004). Synthesis of guanylhydrazones under microwave irradiation. Synthetic Communications 34, 38913899. Moisan, L., Odermatt, S., Gombosuren, N., Carella, A., and Rebek, J., Jr. (2008). Synthesis of an oxazole-pyrrole-piperazine scaffold as an alpha -helix mimetic. European Journal of Organic Chemistry, 1673-1676. Rodriguez, J. M., and Hamilton, A. D. (2006). Intramolecular hydrogen bonding allows simple enaminones to structurally mimic the i, i +4, and i +7 residues of an alpha -helix. Tetrahedron Letters 47, 7443-7446. Shepherd, R. G., and Fellows, C. E. (1948). Iodination of certain phenylsulfonamido and amino heterocycles. Journal of the American Chemical Society 70, 157-160. Volonterio, A., Moisan, L., and Rebek, J., Jr. (2007). Synthesis of pyridazine-based scaffolds as alpha -helix mimetics. Organic Letters 9, 3733-3736.

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130 Watanabe, J.-i., Yokoyama, H., Okada, S., Takaragi, S., Matsuda, H., and Nakanishi, H. (2009). Solid-state polymerization of monophenylbutadiyne derivatives. Molecular Crystals and Liquid Crystals 505, 441-447. Yin, H., Lee, G.-i., Park, H. S., Payne, G. A., Rodriguez, J. M., Sebti, S. M., and Hamilton, A. D. (2005a). Terphenyl-based helical mimetics that disrupt the p53/HDM2 interaction. Angewandte Chemie, International Edition 44, 2704-2707. Yin, H., Lee, G.-i., Sedey, K. A., Kutzki, O., Park, H. S., Orner, B. P., Ernst, J. T., Wang, H. -G., Sebti, S. M., and Hamilton, A. D. (2005b). Terphenyl-Based Bak BH3 alpha Helical Proteomimetics as Low-Molecular-Weight Antagonists of Bcl-xL. Journal of the American Chemical Society 127, 10191-10196.

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131 Chapter Five: Design and Synthesis of Cyclic Urea HIV-1 Protease Inhibitor 5.1 Introduction 5.1.1 AIDS Acquired immune deficiency syndrome (AIDS) was first identified on June 5 th 1981 in the USA (Gottlieb Michael, 2006) This syndrome is caused by the human immunodeficiency virus (HIV), which is believed to have originated in non-human primates in sub-Saharan Africa and then transferred to humans during the 20th century (Gao et al., 1999). While it is hard to define the beginning of the AIDS epidemic, it has Figure 5.1: Estimated number of people living with HIV globally, 1990 2007

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132 been estimated that during the 26 year period between 1981 and 2007 there have been more than 25 million deaths as a result of AIDS infection. This averages out to almost 1 million AIDS related deaths per year. Statistical data shows that the number of people living with HIV is still increasing (Figure 5.1) (WHO, 2009); this implies that the number of deaths caused by AIDS is also increasing. In addition, this virus can be passed from mothers to children during pregnancy or through breast milk (Kallings, 2008). As a result, children are becoming a greater proportion of newly infected AIDS patients each year. 5.1.2 Efforts to treat or cure AIDS Since the discovery of HIV, there have been tremendous efforts made towards targeting this deadly virus. While there are several FDA approved drugs available to help treat this disease, none of them can cure this disease. In addition, those patients living in poor or developing countries, who account for more than 90% of all HIV infected patients, cannot afford the astronomical cost of these therapies (HIV/AIDS programme highlights, WHO report). Thus, designing synthetic routes for novel small molecule HIV inhibitors remains an important goal. This report will focus mainly on the HIV-1 virus, which accounts for about 90% of all HIV infections. 5.1.3 Structure of HIV

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133 HIV belongs to a group of viruses called retro-virus es (Weiss, 1993), which carry their genetic information in the form of RNA. The diagram of a mature HIV virion is shown in Figure 5.2 (OracleThinkQuest). The center of the virion consists of two pieces of the enzymes that are needed for the development of a new virion, such as the reverse transcriptase, integrase, ribonuclease and protease are encoded in these RNA strands. The virion is enclosed in a two layered network. The inner layer is called the conical capsid which is composed of 2000 copies of a viral protein named p24. The outer layer is called the protein matrix and is composed of a viral protein named p17. This kind of multiple-layer enclosure exists to protect the integrity of the virion particles. These layers are then surrounded by the envelope which is an outer membrane that is composed Figure 5.2: Diagram of HIV

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134 of a phospholipid bilayer and is similar to a regular eukaryotic cell membrane. Embedded in the viral envelope are about 70 copies of a viral protein known as Env which is comprised of a cap and a stem. The cap is made from a cluster of three glycoprotein gp120 molecules while the stem is made from a cluster of three glycoproteins gp41 molecules. The function of the Env protein is to identify and bind to 5.1.4 HIV replication mechanism The targets of HIV are mainly the immune cells such as helper T cells (especially CD4 + T cells), macrophages, and dendritic cells. Figure 5.3 (wiki) shows the entire replication process for the creation of a new HIV virion (Brik and Wong, 2003) entry to the host cell is mediated through the interaction between the gp120 protein on the HIV membrane and the CD4 molecule on the target cell. The HIV virion absorbs the the viral envelop with the cell bound to the host cell, the RNA strands and all the viral enzymes are injected into the cell. The viral RNA is then copied into double stranded viral DNA by the reverse enzyme known as integrase. Afterwards, the viral DNA is transcribed into viral mRNA which is then translated into the viral polyprotein. The protease enzyme then helps to finish the maturation process by hydrolyzing the viral polyprotein into individual viral proteins. Finally, the mature viral protein, viral RNA and viral enzymes are packaged membrane to form a new HIV virion.

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135 Figure 5.3: HIV replication cycle 5.1.5 Common targets to inhibit the HIV replication process Studying the infection mechanism has led to the development of several inhibitors, most of which target the viral enzymes. One of the most popular targets at the present time is reverse transcriptase due to the fact that this enzyme does not naturally exist in the human body thus minimizing the side effects of its inhibitors (Zheng et al.,

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136 2005). However, the reverse transcriptase is not actually a good target because of the high replication rate of the virus (10 8 -10 9 virions per day) along with a high error rate of the HIV reverse transcriptase (~1 in 10K bases) which usually lead to a very high resistance to the current drugs On the other hand, another critical enzyme, the protease, has become increasingly targeted because it is critical role for the development of a mature HIV virion. It has been shown that the immature HIV viral polyprotein is not infective (Kohl et al., 1988). 5.1.6 Structure of HIV protease The HIV protease (HIV PR) is believed to belong to the family of aspartic proteases. It contains 99 amino acids which function as a homodimer with only one active site which is C 2 -symmetic when the protein is in the free form (Brik and Wong, 2003). Figure 5.4 shows the cocrystal structure of HIV PR complexed with inhibitor TL-sheet region Figure 5.4: Structure of HIV PR complexed with TL-3

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137 from both of the monomers. The ceiling of the cavity is Ile-50/Ileholds the Asp-25/Asp-25/AspAsp-29/Asp-30/Asphis cavity is very hydrophobic. 5.1.7 HIV protease function mechanism While the mechanism of this proteolysis is not very clear, it is generally accepted that a catalytic water molecule acts as a nucleophile to attack the scissile bond instead of the carboxylic group from Asp-25/Asp(Jaskolski et al., 1991). This then forms a tetrahedral diol intermediate (Figure 5.5). Many potent transition-state inhibitors are designed according to this mechanism. Figure 5.5: Mechanism of HIV PR proteolysis 5.1.8 Cy clic urea as the HIV-1 protease inhibitor In 1997, the DuPont Merck group found that cyclic ureas fit well in the HIV PR active site cavity (Figure 5.6) (De Lucca et al., 1997); however there has been little work

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138 reported since. In this report, we plan to use cyclic urea as the HIV PR inhibitor basic framework and extend the substitutents on it to get better binding affinity. Figure 5.6: X-ray crystal structure of cyclic urea in the active site of HIV PR 5.1.9 Our design strategy We would like to keep the six-membered cyclic urea ring since it sterically fits the active site cavity very well. But we would like to attach more hydrophilic functional groups (e.g. a hydroxyl group on the 4-position) to enhance the hydrogen bonding interaction between our molecule and the Asp-29/Asp-30/AspHIV-1PR. Additional hydrophobic groups on the 5-position should help to increase Van der Waals bonding or hydrophobic interaction between our molecules and the hydrophobic cavity.

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139 In this report, we would also like to develop a general synthetic procedure for making 4-hydroxyl cyclic urea. Eventually, we will be able to create a library of this type of compound in order to test their bioactivities. 5.2 Results and Discussion 5.2.1 Retrosynthesis of 5-hydroxyl cyclic urea The creation of a 5-hydroxyl cyclic urea is challenging because of the instability of the geminal diamino functional group with an adjacent sp 3 carbon center. This kind of functional group is hard to form and easily decomposes. Our strategy in this report is to try to rearrange an even more unstable 1,3-diazetidin-2-one b to achieve our desired compound a (Scheme 5.1). As we can see, the four-membered 1,3-diazetidin-2-one b is highly strained and the nitrogen atom in the ring adjacent to the Boc group is somewhat of a good leaving group. The free amino group in the molecule could selectively open one side of the four-membered ring to form a thermodynamically more stable Scheme 5.1: Retrosynthesis of 5-hydroxyl cyclic urea

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140 six-membered ring a. This four-membered 1,3-diazetidin-2-one b could be made from a 2-oxo-1,3-diazabicyclo[2,2,0]hex-5-ene c molecule through oxidative cleavage of the alkene followed by a Henry reaction. This bicyclic intermediate c could be synthesized by photochemical electrocyclization from pyrimidin-2(1H)-ones d 5.2.2 Synthesis of intermediate 5.3 Following this strategy, the following steps have been carried out (Scheme 5.2): Scheme 5.2: Synthesis of the intermediate 5.3 The first major step of the synthesis is to make a variety of 1,4,6-trisubstituted pyrimidin-2(1H)-ones 5.1 as the photochemical electrocyclization precursors. Table 5.1 shows all the pyrimidinone derivatives that have been synthesized so far. Several procedures have been developed, the most successful one is the condensation between 1,3-disubstituted 1,3-propanedione and N -substituted urea in acetic acid using ptoluenesulfonic acid as the proton source (Katritzky et al., 1982). Additionally, the use of a Dean-Stark arm while refluxing has often been applied. Unfortun at ely, the yield of this step is only poor to fair, e.g. 32% for R 1 = Bn R 2 = i Pr

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141 Table 5.1: Analogs of the synthesized 1,4,6-trisubstituted pyrimidin-2(1H)-ones 5.1 R 1 R 2 Conditions 5.1a Bn i Pr AcOH/PTSA reflux 5.1b Bn Ph AcOH /PTSA reflux 5.1c Bn Me AcOH /PTSA reflux 5.1d Me Me AcOH /PTSA reflux 5.1e Bn H reflux 5.1f Me Ph AcOH /PTSA reflux Using the condensation reaction between 2,6-dimethyl-3,5-heptanedione and benzylurea as a model (Table 5.1 5.1a ), the first step of the cyclization is believed to be the amino group of the N -benzylurea attacking one of the carbonyl groups on 2,6dimethyl-3,5-heptanedione. One of the big problems with this reaction is that it creates a large quantity of side products (Scheme 5.3). This is due to the fact that in these high temperature and acidic reactions conditions, benzylurea is prone to hydrolysis which then generates benzylamine e Benzylamnie then reacts with the protonated benzylurea to form N ,N -dibenzylurea f which then precipitates out of solution thus making this side reaction thermodynamically favorable. Additionally, this side reaction is very fast. Scheme 5.3: Hydrolysis of N -benzylurea under high temperature acidic conditions

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142 Large amounts of precipitate can be seen within 30 minutes while the reaction time of the cyclization is around 36 hours. In fact, pure N ,N -dibenzylurea f can usually be isolated in large amount after the reaction. Due to these facts, the reaction of the diketone is less favored than the reaction of benzylamine with N -benzylurea. Several adjustments have been tried such as increasing the equivalence of benzylurea, decreasing the reaction temperature below 90 C to slow down the formation of benzylamine, increasing the temperature to 150 C to accelerate the cyclization, using solvent free condition, and adding a small amount of dimethylformamide (DMF) to prevent the precipitation. However, no improvement has been made so far. The good thing is that this is the very first step of the synthesis, and the two starting material are readily commercially available. Therefore, the low yield of this reaction is not a bottleneck for the whole synthesis. It is worthwhile to mention that asymmetric diketones have been tried as well in this reaction. However, this procedure lacks regio-selectivity and would yield a mixture of products where the benzyl group on the urea would be found on either side of the parent ring. Also, the two isomers formed here have very similar polarity which makes it impractical to isolate them by chromatography on a large scale. Therefore, no further work has been carried out using asymmetric diketones. 5.2.3 Photochemical electrocyclization of 1,4,6-trisubstituted pyrimidin-2(1H)-ones 5.1

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143 The second step, photochemical electrocyclization of the 1,4,6-trisubstituted pyrimidin-2(1H)-ones to 2-oxo-1,3-diazabicyclo[2.2.0]hex-5-enes, is one of the key steps of the synthesis (Scheme 5.4) (Nishio et al., 1981). While the photo reaction is a very demanding step, it works very efficiently for those substrates which meet the requirements of the reaction. Scheme 5.4: Synthesis of the 1,3-diazetidine-2-one intermediate The light source is the first crucial factor for this reaction to make 5.2. Several lamps such as a Xenon lamp and a mercury vapor lamp (wavelength > 253nm) have been tested, that they could not promote the reaction. We believe the failure of these reactions

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144 is because the photochemical cyclization is an equilibrium reaction; both starting materials and products can absorb photons to go to the reverse of the reaction. The product is a lot less stable than the starting material due to the high ring strain of the bicyclic four-membered rings. Therefore, decomposition of the product is a favorable reaction. One difference between the starting material and the product is that the starting material has a conjugated double bond system while the product does not. This led us to hypothesis that if we could find a light source that can provide photons with a wavelength high enough, it is possible that only the starting material would be activated. Medium pressure mercury lamps meet this requirement. Using a medium pressure mercury lamp with a Pyrex filter provides a sufficient wavelength with a cutoff around 300 nm. This is higher than any of the absorption wavelengths that the product has (Figure 5.7) so the reaction should proceed in a highly favorable manner only towards the products. Figure 5.7: UV absorption of 5.1a

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145 The reaction is also dependent on the substitutents on the pyrimidinone ring. Compounds 5.1d and 5.1f (Table 5.1) have no reaction because those compounds are too polar to be dissolved in benzene, the common solvent for the photo chemical reaction. Compound 5.1e which did not have any solubility issue, did not work because the normal photochemical cyclization is going through a triplet mechanism. The unsubstituted 6-position on the pyrimidinone ring is not able to stabilize this triplet intermediate. Compound 5.1b did not work however, is thought to be because of another reason. For this substrate, when the four-membered product is formed, the double bond in the four-memberded ring would be conjugated with the phenyl group; this would cause the product to absorb photons with higher wavelength. Theoretically, if we can apply a photon generator to generate photons with wavelength in certain ranges, we should be able to get this substrate to work. In summary, pyrimidinones 5.1 with good solubility in benzene (this can be achieved by N-benzyl substitution), and with 6-position substitutuents, activated by photons of proper wavelength (e.g. from medium pressure mercury lamp) should be able to undergo this photochemical cycloaddition. Then we ozonized the double bond to make compound 5.3. Other conditions have been tested, e.g. NaIO 4 /OsO 4 KMnO 4 on solid support; but in both cases, the double bond would not be fully cleaved, and an -hydroxyl ketone 5.8 was isolated as the major product (Scheme 5.5). Scheme 5.5: Side product of the oxidation of 5.2a

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146 It is worthwhile to point out that compound 5.3 is very moisture sensitive. Spectroscopy normally detected both 5.3 and the molecule with one water added to form a five-membered ring (Scheme 5.6). Scheme 5.6: Equilibrium between 5.3 and its water added form turned out to be problematic because the five-membered ring is so stable that regular hydrolysis is not able to cleave the iso butyryl group on the nitrogen atom. We chose to reduce the two new generated C ,O double bond to cleave th at iso butyryl group. However, mild reductants, like sodium borohydride, could only reduce one of double bond, to synthesize compound 5.4. Later, we found that LiBH 4 is able to reductively cleave both of the C ,O double bond to prepare compound 5.5. We also performed Henry reaction on 5.3 to synthesize compound 5.6; the nitro group of 5.6 could then be easily reduced to afford compound 5.7.

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147 5.4 Experimental Procedures of selected compounds 1-Benzyl-4,6-diisopropylpyrimidin-2(1H)-one (5.1a) To a solution of 2,6-dimethyl-3,5-heptanedione (46.7 mmol) in a mixture of acetic acid (18 mL ) and toluene (15 mL) was added N -benzylurea (74.2 mmol) and ptoluenesulfonic acid monohydrate (92.9 mmol). The reaction mixture was stirred under reflux for 12 h. The precipitate was removed by vacuum filtration, and the filtrate was concentrated under reduced pressure. The residue was then extracted between dichloromethane and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product a white solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 3:1) affords 5.1a as a white solid (4.0 g, 32%). 1 H NMR (250 MHz, CDCl 3 ) ppm 1.16 (d, J = 6.77 Hz, 6H), 1.28 (d, J = 6.91 Hz, 6H), 2.83 (m, 1H), 2.95 (m, 1H), 5.38 (s, 2H), 6.15 (s, 1H), 7.11 7.41 (m, 5H). 13 C NMR (75 MHz, CDCl 3 ) ppm 17.4, 17.6, 19.6, 19.9, 29.5, 29.7, 47.4, 82.1, 115.8, 128.1, 128.7, 128.8, 135.6, 160.8, 166.7. 1-Benzyl-4,6-diphenylpyrimidin-2(1H)-one (5.1b)

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148 Compound 5.1b was prepared following the procedure described for 5.1a Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:1) affords 5.1b as a white solid (39%). 1 H NMR (250 MHz, CDCl 3 ) ppm 5.03 (s, 2H), 6.23 (s, 1H), 6.71 7.19 (m, 13H), 8.07 (dd, J = 7.93, 1.68 Hz, 2H). 13 C NMR (75 MHz, CDCl 3 ) ppm 49.6, 102.0, 127.4, 127.6, 128.1, 128.3, 128.5, 128.7, 129.6, 131.6, 134.2, 136.8, 137.5, 157.1, 160.0, 169.5. 1-Benzyl-4,6-dimethylpyrimidin-2(1H)-one (5.1c) Compound 5.1c was prepared following the procedure described for 5.1a Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:1) affords 5.1c as a white solid (45%). 1 H NMR (250 MHz, MeOD) ppm 2.38 (s, 6H), 5.37 (s, 2H), 6.45 (s, 1H), 7.147.44 (m, 5H). 1,4,6-trimethylpyrimidin-2(1H)-one (5.1 d) Compound 5.1d was prepared following the procedure described for 5.1a Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 4:1) affords 5.1c as a

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149 white solid (55%). 1 H NMR (250 MHz, MeOD) ppm 2.57 (s, 3H), 2.72 (s, 3H), 3.70 (s, 3H), 6.84 (s, 1H). 3-Benzyl-4,6-diisopropyl-1,3-diazabicyclo[2.2.0]hex-5en -2-one (5.2a) A solution of 5.1a (3.7 mmol) in 100 mL degassed benzene was irradiated with a medium pressure mercury lamp using a Pyrex filter for 3 days. The reaction mixture was then concentrated under reduced pressure to afford the crude material as a colorless oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:7) affords 5.2a as a colorless oil (0.88 g, 88%). 1 H NMR (250 MHz, CDCl 3 ) ppm 0.92 (d, J = 6.75 Hz, 6H), 1.11 (d, J = 6.91 Hz, 6H), 1.96 2.13 (m, 1H), 2.50 2.64 (m, 1H), 4.40 (dd, J = 93.08, 15.26 Hz, 2H), 5.53 (d, J = 1.62 Hz, 1H), 7.19 7.38 (m, 5H). 13 C NMR (75 MHz, CDCl 3 ) ppm 21.3, 22.4, 30.4, 37.3, 47.8, 99.5, 126.5, 127.8, 129.1, 136.5, 158.2. 3-Benzyl-4,6-dimethyl-1,3-diazabicyclo[2.2.0]hex-5en -2-one (5.2b) Compound 5.2b was prepared following the procedure described for 5.2a Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:7) affords 5.1c as a

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150 colorless oil (72%). 1 H NMR (250 MHz, benzened 6 ) ppm 1.12 (s, 3H), 1.73 (d, J = 1.68 Hz, 3H), 3.98 (dd, J = 63.00, 15.16 Hz, 2H), 5.18 (d, J = 1.68 Hz, 1H), 6.94 7.12 (m, 5H). 1-Benzyl-3-isobutyryl-2-isopropyl-4-oxo-1,3-diazetidine-2-carbaldehyde (5.3) Through a solution of compound 5.2a (0.5g, 1.9 mmol) in 10 mL dichloromethane cooled in acetone/dry ice bath was passed a stream of ozone until the light blue color persisted. The reaction mixture was then flushed with argon (20 min), dimethylsulfide (3 mL) was added, and stirring was continued at room temperature for 2 h. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between DCM and water. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow oil. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:2) affords 5.3 as a colorless oil (0.58 g, >90%). 1 H NMR (250 MHz, CDCl 3 ) ppm 0.84 (d, J = 6.92 Hz, 3H), 0.96 (d, J = 6.85 Hz, 3H), 1.19 (d, J = 6.81 Hz, 3H), 1.25 (d, J = 6.96 Hz, 3H), 2.55 (td, J = 6.80 Hz, 1H), 3.29 (d, J = 6.80 Hz, 1H), 4.56 (dd, J = 48.58, 15.59 Hz, 2H), 7.257.42 (m, 5H). 1-benzyl-4(1 -hydroxy-2-nitroethyl)-3-isobutyryl-4-isopropyl-1,3-diazetidin-2-one (5.6)

PAGE 169

151 To a solution of 5.3 (2.8 mmol) in ice cooled nitromethane (20 mL) was added triethylamine (3.6 mmol). The reaction mixture was stirred at room temperature for overnight. The reaction mixture was then concentrated under reduced pressure. The residue was extracted between ethyl acetate and water. The organic layer was washed with brine, dried over sodium sulfate, and the solvent was removed under reduced pressure to afford the crude product as a yellow solid. Purification by flash column chromatography on silica gel (ethyl acetate/hexane, 1:2) affords 5.3 as a white solid (0.58 g, 52%). 2.4 References Brik, A., and Wong, C.-H. (2003). HIV-1 protease: mechanism and drug discovery. Organic & Biomolecular Chemistry 1, 5-14. De Lucca, G. V., Liang, J., Aldrich, P. E., Calabrese, J., Cordova, B., Klabe, R. M., Rayner, M. M., and Chang, C.-H. (1997). Design, Synthesis, and Evaluation of Tetrahydropyrimidinones as an Example of a General Approach to Nonpeptide HIV Protease Inhibitors. Journal of Medicinal Chemistry 40, 1707-1719. Gao, F., Bailes, E., Robertson, D. L., Chen, Y., Rodenburg, C. M., Michael, S. F., Cummins, L. B., Arthur, L. O., Peeters, M., Shaw, G. M. et al. (1999). Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature (London) 397, 436-441. Gottlieb Michael, S. (2006). Pneumocystis pneumonia -Los Angeles. 1981. In, (United States), pp. 980-981; discussion 982-983.

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152 Jaskolski, M., Tomasselli, A. G., Sawyer, T. K., Staples, D. G., Heinrikson, R. L., Schneider, J., Kent, S. B. H., and Wlodawer, A. (1991). Structure at 2.5-.ANG. resolution of chemically synthesized Human Immunodeficiency Virus Type 1 protease complexed with a hydroxyethylene-based inhibitor. Biochemistry 30, 1600-1609. Kallings, L. O. (2008). The first postmodern pandemic: 25 years of HIV/ AIDS. Journal of internal medicine 263, 218-243. Katritzky, A. R., Salgado, H. J., Chermprapai, A., and Ponkshe, N. K. (1982). Metalation studies with pyrimidines. Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry, 153-158. Kohl, N. E., Emini, E. A., Schleif, W. A., Davis, L. J., Heimbach, J. C., Dixon, R. A. F., Scolnick, E. M., and Sigal, I. S. (1988). Active human immunodeficiency virus protease is required for viral infectivity. Proceedings of the National Academy of Sciences of the United States of America 85, 4686-4690. Nishio, T., Nakajima, N., and Omote, Y. (1981). Photooxygenation of pyrazin-2(1H)ones. Tetrahedron Letters 22, 753-756. OracleThinkQuest http://library.thinkquest.org/03oct/00520/gallery/photos/photo_27.html In. Weiss, R. A. (1993). How does HIV cause AIDS? Science (New York, NY) 260, 12731279. WHO (2009). whqlibdoc.who.int/publications/2010/9789241599450_eng.pdf. In. wiki http://en.wikipedia.org/wiki/File:HIV_gross_cycle_only.png In. Zheng, Y.-H., Lovsin, N., and Peterlin, B. M. (2005). Newly identified host factors modulate HIV replication. Immunology Letters 97 225-234.

PAGE 171

153 Appendix A: Selected 1 H and 13 C NMR Spectra

PAGE 172

154 Methyl 2-(naphthalen-2-yl)acetate (2.1a)

PAGE 173

155 3-Oxo-4-phenylbutanenitrile (2.2a)

PAGE 174

156 es-CO 4-(Naphthalen-1-yl)-3-oxobutanenitrile (2.2b)

PAGE 175

157 4-(Naphthalen-2-yl)-3-oxobutanenitrile (2.2c)

PAGE 176

158 5-Methyl-3-oxohexanenitrile (2.2d)

PAGE 177

159 2-((Dimethylamino)methylene)-3-oxo-4-phenylbutanenitrile (2.3a)

PAGE 178

160 pt-BR 2((D imethylamino)methylene) 4(naphthalen 1yl) 3oxobutanenitrile (2.3b) pt-BR

PAGE 179

161 2((Dimethylamino)methylene)-4-(naphthalen-2-yl)-3-oxobutanenitrile (2.3c)

PAGE 180

162 2((Dimethylamino)methylene)-5-methyl-3-oxohexanenitrile (2.3d)

PAGE 181

163 pt-BR 2((D imethylamino)methylene) 4,4dimethyl 3oxopentanenitrile (2.3e)

PAGE 182

164 4-( tert -Butyl)pyrimidine-5-carbonitrile

PAGE 183

165 4-Benzylpyrimidine-5-carbonitrile (2.4b)

PAGE 184

166 2-Methyl-4-(naphthalen-1-ylmethyl)pyrimidine-5-carbonitrile (2.4c)

PAGE 185

167 4i so -Butyl-2-phenylpyrimidine-5-carbonitrile (2.4d)

PAGE 186

168 4-Benzyl-2-phenylpyrimidine-5-carbonitrile (2.4e)

PAGE 187

169 pt-BR 4tert -Butyl-2-phenylpyrimidine-5ca rbonitrile (2.4f)

PAGE 188

170 4(N aphthalen-1-ylmethyl)-2-phenylpyrimidine-5-carbonitrile (2.4g)

PAGE 189

171 2-Amino-4-benzylpyrimidine-5-carbonitrile (2.4h)

PAGE 190

172 2-Amino-4tert -butylpyrimidine-5-carbonitrile (2.4i)

PAGE 191

173 4-Benzyl-2-methoxypyrimidine-5-carbonitrile (2.4j)

PAGE 192

174 4'-Benzyl-4-(naphthalen-1-ylmethyl)-2'-phenyl-[2,5'-bipyrimidine]-5-carbonitrile (2.6a)

PAGE 193

175 4-Benzyl-4'-(naphthalen-1-ylmethyl)-2'-phenyl-[2,5'-bipyrimidine]-5-carbonitrile (2.6c)

PAGE 194

176 4-Benzyl-2'-methyl-4'-(naphthalen-1-ylmethyl)-[2,5'-bipyrimidine]-5-carbonitrile (2.6d)

PAGE 195

177 4-( tert -Butyl)-2'-methyl-4'-(naphthalen-1-ylmethyl)-[2,5'-bipyrimidine]-5carbonitrile (2.6e)

PAGE 196

178 -Cyano-diisobutyl(1 -naphthylmethyl)-2-phenylterpyrimidine (2.8a)

PAGE 197

179 -Cyano-isobutyl(1 -naphthylmethyl)-4(2 -phenylmethyl)-2phenylterpyrimidine (2.8b)

PAGE 198

180 -Cyano-isobutyl(2 -phenylmethyl)-4(1 -naphthylmethyl)-2phenylterpyrimidine (2.8C)

PAGE 199

181 3-Hydroxy-4-phenylbutanenitrile (3.2)

PAGE 200

182 N',3-Dihydroxy-4-phenylbutanimidamide acetate (3.3)

PAGE 201

183 2(2 -Hydroxy-3-phenylpropyl)-4-isobutylpyrimidine-5-carbonitrile (3.4)

PAGE 202

184 2-Bromo-1-phenylethanone (3.6)

PAGE 203

185 2-Azido-1-phenylethanone (3.7)

PAGE 204

186 2(4 (1 -Hydroxy-3-methylbutyl)-1H-1,2,3-triazol-1-yl)-1-phenylethanone (3.8)

PAGE 205

187 3-(Dimethylamino)-2(4 (1 -hydroxy-3-methylbutyl)-1H-1,2,3-triazol-1-yl)-1phenylprop-2en -1-one (3.9)

PAGE 206

188 1(1 (2 -Amino-4-phenylpyrimidin-5-yl)-1H-1,2,3-triazol-4-yl)-3-methylbutan-1-ol (3.10)

PAGE 207

189 1(1 -(4'iso Butyl-2',4-diphenyl-[2,5'-bipyrimidin]-5-yl)-1H-1,2,3-triazol-4-yl)-3methylbutan-1-ol (3.11)

PAGE 208

190 1-(Dimethylamino)-4-methylpent-1en -3-one (4.1)

PAGE 209

191 4iso -Propylpyrimidin-2-amine (4.2)

PAGE 210

192 5-Iodo-4-isopropylpyrimidin-2-amine (4.3)

PAGE 211

193 4(2 -Amino-4-isopropylpyrimidin-5-yl)-2-methylbut-3-yn-2-ol (4.4)

PAGE 212

194 5(1 -Benzyl-1H-1,2,3-triazol-4-yl)-4-isopropylpyrimidin-2-amine (4.6)B

PAGE 213

195 1-(Dimethylamino)-4-methylhex-1en -3-one (4.9b) 1-cyclopropyl-3-(dimethylamino)prop-2en -1-one (4.9c)

PAGE 214

196 3-(Dimethylamino)-1-phenylprop-2en -1-one (4.9c)

PAGE 215

197 2(2 -Benzylidenehydrazinyl)-4-phenylpyrimidine (4.10)

PAGE 216

198 2-Azido-4-phenylpyrimidine (4.12)

PAGE 217

199 3-Methyl-1(1 (4 -phenylpyrimidin-2-yl)-1H-1,2,3-triazol-4-yl)butan-1-ol (4.13)

PAGE 218

200 4-( tert -Butyl)-2-chloropyrimidine-5-carbonitrile (4.14)

PAGE 219

201 Carbamimidic chloride hydrochloride(4.15)

PAGE 220

202 2-Chloro-4-isobutylpyrimidine-5-carbonitrile (4.16)

PAGE 221

203 4-(Benzyloxy)-2-chloropyrimidine (4.17)

PAGE 222

204 Methyl 2((4 -(benzyloxy)pyrimidin-2-yl)amino)-4-methylpentanoate (4.18)

PAGE 223

205 Methyl 2((4 -(benzyloxy)-5-iodopyrimidin-2-yl)amino)-4-methylpentanoate (4.19)

PAGE 224

206 Methyl 2((4 -(benzyloxy)-5-ethynylpyrimidin-2-yl)amino)-4-methylpentanoate (4.20)

PAGE 225

207 2(4 (4 (B enzyloxy)-2-((1-methoxy-4-methyl-1-oxopentan-2-yl)amino)pyrimidin-5yl)-1H-1,2,3-triazol-1-yl)-3-methylbutanoic acid (4.21)

PAGE 226

208 2-Azido-3-methylbutanoic acid (4.22)

PAGE 227

209 1-Benzyl-4,6-diisopropylpyrimidin-2(1H)-one (5.1a)

PAGE 228

210 1-Benzyl-4,6-diphenylpyrimidin-2(1H)-one (5.1b)

PAGE 229

211 1-Benzyl-4,6-dimethylpyrimidin-2(1H)-one (5.1c) 1,4,6-trimethylpyrimidin-2(1H)-one (5.1d)

PAGE 230

212 3-Benzyl-4,6-diisopropyl-1,3-diazabicyclo[2.2.0]hex-5en -2-one (5.2a)

PAGE 231

213 3-Benzyl-4,6-dimethyl-1,3-diazabicyclo[2.2.0]hex-5en -2-one (5.2b) 1-Benzyl-3-isobutyryl-2-isopropyl-4-oxo-1,3-diazetidine-2-carbaldehyde (5.3)

PAGE 232

214 Appendix B: Selected HRMS

PAGE 233

215 pt-BR 2((D imethylamino)methylene) 4(naphthalen 1yl) 3oxobutanenitrile (2.3b)

PAGE 234

216 2((Dimethylamino)methylene)-4-(naphthalen-2-yl)-3-oxobutanenitrile (2.3c)

PAGE 235

217 4'-Benzyl-4-(naphthalen-1-ylmethyl)-2'-phenyl-[2,5'-bipyrimidine]-5-carbonitrile (2.6a)

PAGE 236

218 -Cyano-4,4 -diisobutyl(1 -naphthylmethyl)-2-phenylterpyrimidine (2.8a)

PAGE 237

219 -Cyano-isobutyl(1 -naphthylmethyl)-4(2 -phenylmethyl)-2phenylterpyrimidine (2.8b ):

PAGE 238

220 N',3-Dihydroxy-4-phenylbutanimidamide acetate (3.3)

PAGE 239

221 2(4 (1 -Hydroxy-3-methylbutyl)-1H-1,2,3-triazol-1-yl)-1-phenylethanone (3.8)

PAGE 240

222 3-(Dimethylamino)-2(4 (1 -hydroxy-3-methylbutyl)-1H-1,2,3-triazol-1-yl)-1phenylprop-2en -1-one (3.9)

PAGE 241

223 1(1 (2 -Amino-4-phenylpyrimidin-5-yl)-1H-1,2,3-triazol-4-yl)-3-methylbutan-1-ol (3.10)

PAGE 242

224 1(1 -(4'iso Butyl-2',4-diphenyl-[2,5'-bipyrimidin]-5-yl)-1H-1,2,3-triazol-4-yl)-3methylbutan-1-ol(3.11)

PAGE 243

225 5-Iodo-4-isopropylpyrimidin-2-amine (4.3)

PAGE 244

226 4(2 -Amino-4-isopropylpyrimidin-5-yl)-2-methylbut-3-yn-2-ol (4.4)

PAGE 245

227 2-Azido-4-phenylpyrimidine (4.12)

PAGE 246

228 3-Methyl-1(1 (4 -phenylpyrimidin-2-yl)-1H-1,2,3-triazol-4-yl)butan-1-ol (4.13)

PAGE 247

229 Methyl 2((4 -(benzyloxy)pyrimidin-2-yl)amino)-4-methylpentanoate (4.18)

PAGE 248

230 Methyl 2((4 -(benzyloxy)-5-ethynylpyrimidin-2-yl)amino)-4-methylpentanoate (4.20)

PAGE 249

231 2(4 (4 -(Benzyloxy)-2-((1-methoxy-4-methyl-1-oxopentan-2-yl)amino)pyrimidin-5yl)-1H-1,2,3-triazol-1-yl)-3-methylbutanoic acid (4.21)

PAGE 250

232 Appendix C : X-ray Crystallographic Data

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A bout the Author Mingzhou Zhou is currently a research assistant at the H. Lee Moffitt Cancer & Research Institute at the University of South Florida. He is pursuing his PhD with Prof. Mark L. McLaughlin from research in design and synthesis of non peptidic helical mimetics and HIV 1 protease inhibitors. Prior to that, h e received his BS degree in chemistry at University of Science and Technology of C hina.


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Zhou, Mingzhou.
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Design and combinatorial synthesis approach of non-peptidic trimeric small molecules mimicking i, i + 4(3), i + 7 positions of alpha-helices
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Dissertation (PHD)--University of South Florida, 2010.
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ABSTRACT: Protein-protein interactions are key to several biological processes that facilitate signal transduction and many other processes. These interactions are involved in pathways that are critical to many human diseases. Targeting specific protein-protein interactions is a challenging goal because protein-protein interactions are predominately through hydrophobic interactions. Antagonists of the protein-protein interactions need to be perfectly fit into the binding pockets to ensure the activity. The alpha-helical domain of the proteins behaves as the recognition motifs for numerous protein-protein, and protein-nucleic acid interactions. Research has shown that pathways of many diseases contain protein-protein interactions involving alpha-helical domains, e.g. neurological disorders, bacterial infections, HIV and cancer, etc. It is difficult yet very important to design small molecules to target the shallow binding areas of protein-protein interactions. So far the most successful one is Hamilton's 1,4-terphenylene scaffold, which has been used to target the interactions between p53/MDM2, Bak/Bcl-xL etc. Inspired by this, we designed and synthesized three new scaffolds of non-pepditic alpha-helical mimetics, mimicking the i, i + 4, i + 7 positions of an alpha-helix. There are three basic principles that were leading our design. The side chains of our designed molecules should act as mimetics of the side chains of an alpha-helix. Second, our molecules should possess improved water solubility. Third, the molecules should be easy to synthesize to generate a focused library. Some of our molecules, including the ones whose molecular weight are as low as 294, started to show some inhibition against p53/MDM2 interactions.
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